Compositions and methods utilizing nitroxides in combination with biocompatible macromolecules

ABSTRACT

Compositions and processes to alleviate free radical toxicity are disclosed based on the use of nitroxides in association with physiologically compatible macromolecules. In particular, hemoglobin-based red cell substitutes are described featuring stable nitroxide free radicals for use in cell-free hemoglobin solutions, encapsulated hemoglobin solutions, stabilized hemoglobin solutions, polymerized hemoglobin solutions, conjugated hemoglobin solutions, nitroxide-labelled albumin, and nitroxide-labelled immunoglobulin. Formulations are described herein that interact with free radicals, acting as antioxidant enzyme-mimics, which preserve nitroxides in their active form in vivo. Applications are described including blood substitutes, radioprotective agents, imaging agents, agents to protect against ischemia and reperfusion injury, and in vivo enzyme mimics among others.

This is a continuation-in-part of co-pending application Ser. No.08/417,132, filed Mar. 31, 1995, which is a continuation-in-part ofapplication Ser. No. 08/291,590, filed Aug. 15, 1994, now U.S. Pat. No.5,591,710 which is a continuation-in-part of application Ser. No.08/107,543, filed on Aug. 16, 1993 now abandoned.

FIELD OF THE INVENTION

This invention relates to the combined use of membrane permeablenitroxide and membrane impermeable nitroxide in includingnitroxide-labelled macromolecules, including polypeptide e.g.,hemoglobin, albumin, immunoglobulins, polysacharide, e.g., dextran,hydroxylethyl starch and artificial membranes, to alleviate the toxiceffects of oxygen-related species in a living organism and for thediagnosis and treatment of certain physiological conditions. Thisinvention also relates to nitroxides and nitroxide-labelledmacromolecules used in combination with low molecular weight nitroxidessustain the in vivo effect of the nitroxide. This invention alsodiscloses novel compounds and methods featuring nitroxides used incombination with physiologically compatible cell-free and encapsulatedhemoglobin solutions for use as a red cell substitute. Additionally,this invention describes the above nitroxides in combination with otherphysiologically active compounds, including other nitroxides, to protectfrom pathological damage and oxidative stress caused by free radicalsand describes their use in diagnosis and in the treatment of disease.

BACKGROUND OF THE INVENTION

Although the physiological mechanisms of oxygen metabolism have beenknown for many years, an understanding of the role played by oxidativestress in physiology and medicine is not completely understood. Theimpact of oxygen-derived free radicals on physiology and disease is atopic of increasing importance in medicine and biology. It is known thatdisease and injury can lead to levels of free radicals which far exceedthe body's natural antioxidant capacity--the result is oxidative stress.Oxidative stress is the physiological manifestation of uncontrolledtoxic free radicals, most notably that result from toxic oxygen-relatedspecies. Toxic free radicals are implicated as a causative factor inmany pathologic states, including ischemia-reperfusion injury, shock,alopecia, sepsis, certain drug toxicities, toxicities resulting fromoxygen therapy in the treatment of pulmonary disease, clinical oraccidental exposure to ionizing radiation, trauma, closed head injury,burns, psoriasis, in the aging process, and many others.

Therefore, a need exists for compositions and methods which detoxifyfree radicals and related toxic species and which are sufficientlyactive and persistent in the body to avoid being rapidly consumed whichincreases in free radical concentrations are encountered.

Furthermore, evidence has been developed which demonstrates that freeradicals contribute to aggravate a number of other disease statesincluding cancer, ulcers and other gastro-intestinal conditions,cataracts, closed head injury, injury to the nervous system, andcardiovascular disease to name a few. As a result of their highreactivity, free radicals can oxidize nucleic acids, biologicalmembranes, and other cell components, resulting in severe or lethalcellular damage, mutagenesis, or carcinogenesis. Anti-cancerradiotherapy, as well as a number of antitumor drugs, act by generatingfree radicals which are toxic to tumor cells, but are also toxic tonormal cells which are exposed during cell division causing theundesirable side-effects of cancer therapy. Indeed, it is believed thatmany pathologic processes have as their common final pathway thegeneration of free radicals which are the direct or a substantialcontributing cause of the observed pathology. As the importance ofoxidative stress in living systems becomes appreciated, a continuingneed exists for compounds and methods that can function as anti-oxidantsand which can be designed to interact with oxygen-derived free radicalsto alleviate their toxicity in biological systems, particularly inhumans. In other applications, the toxic free radicals may be coincidentto a beneficial treatment such as radiation administered as part ofcancer radiotherapy. For example, since the mechanism by which ionizingradiation causes physiologic damage to an organism involves, at least inpart, a free radical interaction with cells, compounds which possess orinteract with free radicals exhibit a localized effect on tissuesexposed to radiation therapy controlling collateral damage caused by atherapeutical treatment. Additionally, apart from any clinicallysignificant function, since the unpaired electrons in free radicalsspecies are detectable by spectroscopy, free radical reactions may bemonitored in vivo and compounds which interact with free radicals areobservable by spectroscopic techniques.

Several therapeutic approaches have been proposed to reduce pathologiclevels of free radicals. Ideally, safe and effective antioxidant agentswould augment the patient's antioxidant capacity and assist in blockingmany pathologic free-radical based toxicities at the stage of freeradical generation. However, the development of methods and compounds tocombat oxidative stress or the toxicity associated with oxygen-relatedspecies has enjoyed limited success. The usefulness of manyanti-oxidants is limited by short duration of action in vivo, toxicityat effective dosage levels, the inability of many compounds to crosscell membranes, and an inability to counter the effects of high levelsof free radicals. For example, the administration of the enzymesuperoxide dismutase (SOD) or catalase can promote the conversion oftoxic free radical related species to a non-toxic form. However, theseenzymes do not function effectively in the intracellular space.Procysteine as a GSH precursor, as well as vitamins and otherantioxidant chemicals, can enhance the body's natural antioxidantcapacity, but are unable to deal with the higher levels of free radicalsencountered in injury and disease and are rapidly consumed by the body.

Free radical species are notoriously reactive and short-lived. Suchreactivity is a particularly serious hazard in biological systemsbecause detrimental chemical reactions between a free radical and bodytissue occurs in very close proximity to the site where the free radialis generated. Therefore, compounds which inherently function to reducefree radical concentrations have some beneficial effect, although theeffect may not be clinically significant unless the therapeutic effectcan be concentrated and localized in a particular region of the bodysuch as the cardiovascular system or in discrete tissue such as the siteof radiation administration.

The difficulties encountered in creating a blood substitute suitable forlarge volume intravenous administration are an acute example of thedifficulty in preventing or alleviating systemic toxicity caused byoxygen-related species. Scientists and physicians have struggled fordecades to produce a blood substitute that could be safely transfusedinto humans. Persistent blood shortages and the problems of incompatibleblood types, cross-matching, and the communication of disease have ledto a broad-based effort by private industry, universities, andgovernments to discover a formulation that would allow a large volume ofa blood substitute to be safely transfused without significantphysiological side effects. At present, several companies are conductingclinical trials on experimental blood substitutes. However, adversephysiological reactions and the inherent complexity of the research anddevelopment process have impeded progress through the regulatoryapproval stages and have impeded the development of a clinically usefulblood substitute.

A Research Advisory Committee of the United States Navy issued a reportin August 1992 outlining the efforts by several groups to produce ablood substitute, assessing the status of those efforts, and generallydescribing the toxicity problems encountered. The Naval ResearchAdvisory Committee Report reflects the current consensus in thescientific community that even though the existing blood substituteproducts, often termed "hemoglobin-based oxygen carriers" (HBOC), havedemonstrated efficacy in oxygen transport, certain toxicity issues areunresolved. The adverse transfusion reactions that have been observed inclinical studies of existing hemoglobin-based oxygen carriers (HBOC)include systemic hypertension and vasoconstriction. These adversereactions have forced a number of pharmaceutical companies to abandontheir clinical trials or to proceed at low dosage levels.

Solving the toxicity problem in the existing hemoglobin-based bloodsubstitutes has been given a high priority by the United StatesGovernment. A Naval Research Committee recommendation has beenimplemented by the National Institute of Health in the form of a RequestFor Proposal (PA-93-23) on the subject of "Hemoglobin-Based OxygenCarriers: Mechanism of Toxicity." Therefore, the medical and scientificcommunity suffers from an acute and pressing need for a blood substitutethat may be infused without the side effects observed with the existinghemoglobin-based oxygen carriers.

The red blood cells are the major component of blood and contain thebody's oxygen transport system. It has long been recognized that themost important characteristic of a blood substitute is the ability tocarry oxygen. The red blood cells are able to carry oxygen because theprimary component of the red cells is hemoglobin, which functions as theoxygen carrier. Most of the products undergoing clinical testing asblood substitutes contain hemoglobin that has been separated from thered blood cell membranes and the remaining constituents of the red bloodcells and has been purified to remove essentially all contaminants.However, when hemoglobin is removed from the red cells and placed insolution in its native form, it is unstable and rapidly dissociates intoits constituent subunits. For this reason, the hemoglobin used in ahemoglobin-based oxygen carrier (HBOC) must be stabilized to preventdissociation in solution. Substantial expenditures in scientific laborand capital were necessary to develop hemoglobin-based products that arestable in solution, and which are stabilized in such a way that theoxygen transport function is not impaired. The ability of the existinghemoglobin-based oxygen carriers to transport oxygen has been wellestablished (See U.S. Pat. Nos. 3,925,344; 4,001,200; 4,001,401;4,053,590; 4,061,736; 4,136,093; 4,301,144; 4,336,248; 4,376,095;4,377,512; 4,401,652; 4,473,494; 4,473,496; 4,600,531; 4,584,130;4,857,636; 4,826,811; 4,911,929 and 5,061,688).

In the body, hemoglobin in the red cells binds oxygen molecules as theblood passes through the lungs and delivers the oxygen moleculesthroughout the body to meet the demands of the body's normal metabolicfunction. However, the atmospheric oxygen that most living beings mustbreathe to survive is a scientific and medical paradox. On the one hand,almost all living organisms require oxygen for life. On the other hand,a variety of toxic oxygen-related chemical species are produced duringnormal oxygen metabolism.

With respect to oxidative stress resulting from the transportation ofoxygen by hemoglobin, it is known that in the process of transportingoxygen, the hemoglobin (Hb) molecule can itself be oxidized by theoxygen (O₂) molecule it is carrying. This auto-oxidation reactionproduces two undesirable products: met-hemoglobin (met-Hb) and thesuperoxide anion (·O₂ ⁻). The chemical reaction may be written asfollows:

    Hb+4O.sub.2 →met-Hb+4·O.sub.2.sup.-          1!

The superoxide anion (·O₂ ⁻) is an oxygen molecule that carries anadditional electron and a negative charge. The superoxide anion ishighly reactive and toxic.

As described in detail herein, free radical species, such as thesuperoxide anion are implicated as the agents of cell damage in a widerange of pathological processes. In the case of oxygen transport byhemoglobin, potentially damaging oxidative stress originates with thesuperoxide anion being generated by the auto-oxidation of hemoglobin andresults from the subsequent conversion of the superoxide anion to toxichydrogen peroxide in the presence of the enzyme superoxide dismutase(SOD) by the following reaction: ##STR1## The reaction whereby a freeradical species generates toxic chemical species in vivo or causescellular damage is seen repeatedly in pathologic conditions whereoxidative stress is a factor. The presence of the superoxide anion andhydrogen peroxide in the red blood cells is believed to be the majorsource of oxidative stress to the red cells.

Apart from oxygen transport by the hemoglobin contained therein, a lessrecognized characteristic of the red cells is that they contain aspecific set of enzymes which are capable of detoxifying oxygen-relatedchemical species produced as by-products of oxygen metabolism. Withoutthe protection of these specific enzyme systems, autoxidation ofhemoglobin would lead to deterioration and destruction of the red cells.In the body, however, the reserve capacity of the enzyme systems in thered cells protects the body from oxygen toxicity by converting thesuperoxide anion generated during normal metabolism to non-toxic speciesand thereby controls the level of oxidative stress. However, if thisenzyme system breaks down, the integrity of the red cells will bedamaged. A lesion of the gene that produces one of the enzymes in theprotective system in the red blood cells will cause an observablepathological condition. For example, glucose-6-phosphate dehydrogenasedeficiency, a genetic disorder of red cells, is responsible for hydrogenperoxide induced hemolytic anemia. This disorder is due to the inabilityof the affected cells to maintain NAD(P)H levels sufficient for thereduction of oxidized glutathione resulting in inadequate detoxificationof hydrogen peroxide through glutathione peroxidase (P. Hochstein, FreeRadical Biology & Medicine, 5:387 (1988)).

The protective enzyme system of the red blood cells converts the toxicsuperoxide anion molecule to a non-toxic form in a two-step chemicalpathway. The first step of the pathway is the conversion of thesuperoxide anion to hydrogen peroxide by the enzyme superoxide dismutase(SOD) (See Equation 2!). Because hydrogen peroxide is also toxic tocells, the red cells contain another enzyme, catalase, which convertshydrogen peroxide to water as the second step of the pathway (SeeEquation 3!). ##STR2## Red cells are also capable of detoxifyinghydrogen peroxide and other toxic organoperoxides using the enzymeglutathione peroxidase which reacts with glutathione to convert hydrogenperoxide and organoperoxides to water. Red cells also contain an enzymeto prevent the build up of the met-hemoglobin produced by theauto-oxidation of hemoglobin. The enzyme met-hemoglobin reductaseconverts met-hemoglobin back to the native form of hemoglobin.Therefore, in the body, the toxic effects of the auto-oxidation ofhemoglobin are prevented by specific enzyme-based reaction pathways thateliminate the unwanted by-products of oxygen metabolism.

The enzymatic oxygen detoxification functions of superoxide dismutase,catalase, and glutathione peroxidase that protect red blood cells fromoxygen toxicity during normal oxygen transport do not exist in thehemoglobin-based oxygen carriers (HBOC) developed to date. Without theoxygen detoxification function, the safety of the existing HBOCsolutions will suffer due to the presence of toxic oxygen-relatedspecies.

The principle method by which the existing HBOC solutions aremanufactured is through the removal of hemoglobin from the red cells andsubsequent purification to remove all non-hemoglobin proteins and otherimpurities that may cause an adverse reaction during transfusion (SeeU.S. Pat. Nos. 4,780,210; 4,831,012; and 4,925,574). The substantialdestruction or removal of the oxygen detoxification enzyme systems is anunavoidable result of the existing isolation and purification processesthat yield the purified hemoglobin used in most HBOC. Alternatively,instead of isolating and purifying hemoglobin from red cells, purehemoglobin has been produced using recombinant techniques. However,recombinant human hemoglobin is also highly purified and does notcontain the oxygen detoxification systems found in the red cells. Thus,the development of sophisticated techniques to create a highly purifiedhemoglobin solution is a mixed blessing because the purificationprocesses remove the detrimental impurities and the beneficial oxygendetoxification enzymes normally present in the red cells and ultimatelycontributes to oxygen-related toxicity.

One of the observed toxic side effects resulting from intravenousadministration of the existing HBOCs is vasoconstriction orhypertension. It is well known that the enzyme superoxide dismutase(SOD) in vitro will rapidly scavenge the superoxide anion and prolongthe vasorelaxant effect of nitric oxide (NO). Nitric oxide is a moleculethat has recently been discovered to be the substance previously knownonly as the "endothelium-derived relaxing factor" (EDRF). Theprolongation of the vasorelaxant effect of nitric oxide by SOD has beenascribed to the ability of SOD to prevent the reaction between thesuperoxide anion and nitric oxide. (M. E. Murphy et. al., Proc. Natl.Acad. Sci. USA 88:10860 (1991); Ignarro et.al. J. Pharmacol. Exp. Ther.244: 81 (1988); Rubanyi Am. J. Physiol. 250: H822 (1986); Gryglewskiet.al. Nature 320:454 (1986)).

However, in vivo, the inactivation of EDRF by the superoxide anion hasnot been observed and is generally not thought to be likely.Nevertheless, certain pathophysiological conditions that impair SODactivity could result in toxic effects caused by the superoxide anion(Ignarro L. J. Annu. Rev. Pharmacol. Toxicol. 30:535 (1990)). Thehypertensive effect observed in preclinical animal studies of theexisting HBOC solutions suggests that the concentration of superoxideanion in large volume transfusions of the existing HBOCs is the causefor the destruction of EDRF and the observed vasoconstriction andsystemic hypertension.

It is, therefore, important to delineate the hypertensive effectresulting from the reaction of the superoxide anion with nitric oxide(NO) from that resulting from extravasation and the binding of NO byhemoglobin. Upon transfusion of an HBOC, the hemoglobin can also depressthe vasorelaxant action of nitric oxide by reacting with nitric oxide toyield the corresponding nitrosyl-heme (NO-heme) adduct. In particular,deoxy-hemoglobin is known to bind nitric oxide with an affinity which isseveral orders of magnitude higher than that of carbon monoxide.

These hemoglobin-NO interactions have been used to assay for nitricoxide and to study the biological activity of nitric oxide. For example,the antagonism of the vasorelaxant effect of nitric oxide by hemoglobinappears to be dependent on the cell membrane permeability of hemoglobin.In intact platelets, hemoglobin did not reverse the effect of L-argininewhich is the precursor of nitric oxide. In contrast, in the cytosol oflysed platelets, hemoglobin is the most effective inhibitor ofL-arginine induced cyclic-GMP formation mediated by nitric oxide. Theseexperiments demonstrated that the hemoglobin did not penetrate theplatelet membrane effectively. (Radomski et al., Br. J. Pharmacol.101:325 (1990)). Therefore, one of the desired characteristics of theHBOCs is to eliminate the interaction of nitric oxide with hemoglobin.

Hemoglobin is also known to antagonize both endotheliumdependentvascular relaxation (Martin W. et. al. J. Pharmacol. Exp. Ther. 232:708(1985)) as well as NO-elicited vascular smooth muscle relaxation(Grueter C. A. et al., J. Cyclic. Nucleotide Res. 5:211 (1979)).Attempts have been made to limit the extravasation and hypertensiveeffect of hemoglobin by chemically stabilizing, polymerizing,encapsulating, or conjugating the hemoglobin in the HBOCs to prolong thecirculation time. Therefore, although the current HBOCs are relativelymembrane impermeable and able to transport oxygen, the HBOC solutions donot have the capability of preventing the reaction between superoxideanion and nitric oxide when transfused. The above example demonstratesthe difficulty in addressing the oxygen toxicity/stress issue, evenwhere the reactions mechanisms of oxygen transport are reasonably wellunderstood, and despite decades of research to improve the hemoglobinproduction and formulation process.

An ideal solution to the toxicity problems of the existing bloodsubstitutes would be a hemoglobin-based formulation that combines theoxygen-transport function of the existing HBOCs with the oxygendetoxification function of the red cells. However, a simple addition ofthe enzyme superoxide dismutase (SOD) into an existing HBOC solutionwould not be desirable because, by reducing the concentration ofsuperoxide anion, the reaction whereby hemoglobin is oxidized tomet-hemoglobin would be encouraged, leading to an undesirable build-upof met-hemoglobin (See Equation 1!). Also, it is not desirable toencourage the conversion of the superoxide anion to hydrogen peroxide ina hemoglobin solution because the hydrogen peroxide is toxic andreactive and will decompose to toxic hydroxyl radicals or form othertoxic organoperoxides during storage.

Because synthetic blood substitutes would ideally be infusible in largequantities, compounds which interact with free radicals must be able tooffer sustained in vivo function and be stable and non-toxic. Pursuantto this invention, nitroxides and nitroxide-labelled macromolecules,including hemoglobin, albumin and others are used to alleviate the toxiceffects of free radical species in a living organism.

The capability of nitroxides, used together with biologicalmacromolecules pursuant to this invention, to control the damage causedby free radicals in vivo creates the ability to design therapeutic anddiagnostic nitroxide-containing formulations and methods for their usewhich have a broad range of applications. A large number ofphysiological states and processes where oxygen-derived free radicalsare present may be treated or diagnosed by the use of the compoundsdescribed herein. The use of membrane-permeable, low molecular weightnitroxides in combination with biocompatible macromolecules such ashemoglobin, albumin, and others, allows the researcher to tailor thenitroxide-containing formulation to fit the specific environment ofinterest.

A multi-component nitroxide-based system also functions as aradioprotective agent for use in cancer radiotherapy and in thetreatment of radiation exposure. In clinical applications, the efficacyof radiation therapy will be enhanced by allowing higher radiationdosages to be used safely.

There has long been a need for agents which can protect against the illeffects of ionizing radiation encountered in the course of medicalradiotherapy or as the result of environmental radiation exposure. Suchagents would also be useful tools in research on mechanisms of radiationcytotoxicity. Cysteamine, a sulfur-containing compound, was one of theearliest radioprotective agents identified. Its discovery prompted theUnited States Department of Defense to sponsor the synthesis andsystematic screening of over 40,000 compounds in an attempt to find moreeffective agents. This monumental undertaking resulted in the discoveryof a few radiation protectors such as the aminothiol compound known asWR-2721. More recently superoxide dismutase, interleukin I, andgranulocyte-macrophage colony-stimulating factor have been shown to haveradioprotectant activity. In a comparison of these agents, WR-2721showed the most substantial and selective protection of normal tissues.However, when used in patients undergoing cancer radiotherapy, concernover inherent toxicity and nonselective protection of tumor dampenedenthusiasm for the use of WR-2721.

Certain stable nitroxides have been found to have antioxidant andradioprotectant activities. However, membrane permeable nitroxides arerapidly reduced in vivo to an inactive form and may be toxic in elevateddoses. The utility of administration of membrane permeable nitroxidescan be substantially enhanced pursuant to this invention.

SUMMARY OF THE INVENTION

This invention discloses stable nitroxides used in connection withbiologically compatible macromolecules including other nitroxides fortherapy and diagnosis in biological systems. In particular, thisinvention describes low molecular weight, membrane-permeable nitroxidesused in connection with nitroxides bound in a high molar ratio tobiocompatible macromolecules such as albumin and hemoglobin. In certainapplications, an interaction between one form of a nitroxide and anotherform of nitroxide with a differential free radical stability facilitateselectron or spin transfer between the species. The differentialstability may result from the electrochemical environment of the speciesor from the inherent nature of the compound. This invention alsocontemplates the use of stable nitroxide free radicals, precursors andderivatives, hereafter referred to collectively as "nitroxide(s)", toprovide the oxygen detoxification function of the red cells tohemoglobin-based blood substitutes and to alleviate oxidative stress andto avoid biological damage associated with free radical toxicity,including inflammation, radiation, head injury, shock, post-ischemicreperfusion injury, ionizing radiation, alopecia, cataracts, sepsis,ulcers, and the aging process, among others.

In certain embodiments, stable nitroxides or derivatives thereof areused to create several formulations for a blood substitute that willpossess the oxygen detoxification function of the red cells. Theseformulations may be described herein as hemoglobin-based red cellsubstitutes (HRCS) because the oxygen transport capability of thehemoglobin-based oxygen carriers (HBOC) is enhanced by providing theoxygen detoxification function of the body's red cells. This permits thedesign of vasoneutral hemoglobin-based oxygen carriers which avoid thehypertension observed in many HBOC.

To overcome the drawbacks in the use of nitroxides alone, in preferredembodiments of this invention, a polynitroxide-labelled macromolecule,such as Tempo-labelled human serum albumin is infused together with afree membrane-permeable nitroxide to provide extended activity of thenitroxide in vivo. One benefit of such a formulation is an improvedradioprotective agent which can be used in both diagnostic andtherapeutic medical application and to protect against exposure toradiation from any source. In therapeutic medical applications,increased dosages of radiation are enabled to be administered therebyimproving the possibility that radiation therapy will be successful.This capability is particularly significant in certain tumors such asthose in the brain, and, is useful in combination with imaging andoxygen delivery as described herein, particularly with those tumorscontaining regions of hypoxia.

Also, nitroxides are detectable by electron paramagnetic resonancespectroscopy and nuclear magnetic resonance spectroscopy. With thedevelopment of advanced imaging instrumentation, images of intactbiological tissues and organs are available based on a measurement anddetection of the stable free radical of a nitroxide. Pursuant to thisinvention, active nitroxide levels in the body may be maintained for aprolonged period of time allowing both improved image contrast andlonger signal persistence than seen with low molecular weight membranepermeable nitroxides alone. Moreover, unlike certain existingimage-enhancing agents, the compositions disclosed here are capable,ofcrossing the blood-brain barrier.

Additionally, due to their antioxidant activity, the compositionsdisclosed herein have therapeutic value which, in combination with theirdiagnostic value, allows the novel compositions and methods of thisinvention to be used advantageously in a wide variety of applications.

Materials and methods are also described for the preparation andadministration of stable nitroxides in several forms. In particular,inactive, relatively non-toxic precursors or derivatives ofmembrane-permeable nitroxides are described which are converted in vivoby other compounds described herein to biologically active nitroxides,or antioxidant enzyme mimics. In either case, the chemically reduced(inactive) nitroxide may be reactivated, by other nitroxides ofdifferential stability or by nitroxide-labelled macromolecular species,after having been reduced in the process of detoxifying harmful freeradicals. As a result of this regeneration effect, the nitroxides ofthis invention have longer half lives in vivo compared to low molecularweight, membrane-permeable nitroxides alone. Thus, this inventionprovides compositions and methods to enhance the effectiveness of anyapplication where nitroxides are efficacious.

Using the multi-component system of this invention, a dynamicequilibrium is created between low molecular weight, membrane-permeablenitroxides and membrane permeable nitroxide-containing species ofdifferential stability. In particular, a nitroxide-based compoundfeaturing a nitroxyl group capable of accepting an electron from anothernitroxide, such as a membrane impermeable macromolecular-bound nitroxidecapable of accepting an electron from the hydroxylamine derivative, mayact as an enzyme mimic to regenerate the active function of the membranepermeable nitroxides, or vice versa as an electron acceptor, to convertthe hydroxylamine form of a nitroxide to the free radical form.

The capability to maintain the concentration of an active nitroxide invivo pursuant to this invention offers advantages in virtually anyapplication where administration of a nitroxide is beneficial but theutility is limited, due to rapid reduction in vivo or where theoptimally effective dose of a membrane preamble nitroxide is toxic. Forexample, the increased active half-life of nitroxide in vivo pursuant tothis invention provides radiation protection and improved imaging inclinical and other applications where the effective dose of a lowmolecular weight membrane permeable nitroxide is toxic or rapidlyconsumed.

Nitroxides, which are paramagnetic by virtue of a stable unpairedelectron, function as imaging agents in nuclear magnetic resonanceimaging (NMR/MRI) and in electron paramagnetic resonance imaging(EPR/ERI). However, due to the rapid reduction of nitroxide to aspectroscopically invisible species, most typically the hydroxylamineform, the utility of such agents is limited. Because free radicalspecies are implicated in reperfusion injury, and are known to accompanyoxygen metabolism, ischemic tissue injury, and hypoxia may be observedusing the compositions of this invention as imaging agents.Additionally, the antioxidant, enzyme-mimic effect of the compositionsof this invention provides the added benefit of protection fromoxidative damage.

A distinct advantage of the multi-component nitroxide based system isthe capability to deliver the antioxidant, radioprotective,anti-ischemic, image-enhancing, enzyme-mimic, etc. function to severalregions of the body, such as the vascular compartment, interstitialspace, and intracellular regions or to a particular region based onselective permeability of the biological structure or utilizing knownmethods of administration which provide targeted or localized effect.The researcher or clinician can tailor the multi-component systemdescribed here to fit the application. For example, differentformulations described herein have differing levels of vasorelaxanteffect. The ability to tailor the selection of the nitroxide-containingspecies of the multi-component system of the invention provides theability to selectively treat or diagnose particular disease states orconditions or to provide increases or decreases in the free radical formof the nitroxide. For example, as will be appreciated by those skilledin the art, the invention can be particularly applied to thecardiovascular system by intravenous of one or more of the components ofthe multi-component system described herein. Similarly, a particularregion of the skin may be selected by topical administration of onenitroxide while administering the other species by topical, oral, orintravenous administration depending on the particular application ofthis invention.

Fundamentally, in certain embodiments, a nitroxide (including precursorsand metabolic substrates) is provided which is selected to perform thedesired function, i.e., radioprotection, imaging, enzyme-mimic, etc.,and another nitroxide-based species is provided as a reservoir ofactivity. In terms of electron spin transfer, one species may beconsidered an "acceptor" nitroxide and the other a "donor nitroxide." Incertain embodiments, the "donor" and "acceptor" may remain substantiallyphysically separated in vivo and should have different stabilities intheir free radical moieties. In a preferred embodiment, the acceptornitroxide is a polynitroxide albumin which distributes predominantly inthe vascular space and acts as a storehouse of activity. The donorspecies is typically a low-molecular weight, membrane permeable speciessuch as TPL or TPH. Alternatively, the donor species may be membraneimpermeable and the acceptor species membrane permeable and the speciesselected such that the activity of a nitroxide is inhibited.

Those of ordinary skill will appreciate that the individual speciesselected as the donor or acceptor may vary as long as substantialphysical separation is maintained and differential stability isachieved. For example, the same nitroxide species may act as bothacceptor and donor. In such an example, TPL labelled at a number ofamino groups on a macromolecular species such as albumin provides asubstantially membrane-impermeable acceptor nitroxide. Differentialstability of the macromolecular-bound TPL is provided by labelling atthe amino groups such that the remaining carboxyl groups create anacidic microenvironment yielding a less stable free radical state in thealbumin-bound TPL. Alternatively, different unbound nitroxide speciesmay be provided which, by virtue of their inherent chemical andelectrical structure, provide the requisite separation and differentialstability.

The dynamic equilibrium which is created by the compounds of thisinvention is between a reduced form of a nitroxide and an oxidized formsuch that one is active in vivo and the other inactive. The fundamentalmechanism is acceptance of an electron from a first nitroxide,particularly the reduced hydroxylamine derivative thereof, by thenitroxyl group of a second nitroxide. The second nitroxide is capable ofaccepting an electron when it contacts the first nitroxide by virtue ofthe differential stability of the free radical nitroxyl group.In oneexample, the free radical or "oxidized" form, e.g. TPL, becomes rapidlyreduced to TPH until regenerated to TPL by polynitroxide albumin (PNA).##STR3##

The preferred compositions using nitroxides in connection withbiocompatible macromolecules may be varied; for example, with aphysiologically compatible solution for infusion such as ahemoglobin-based oxygen carrier, the compositions include: 1)nitroxide-containing compounds added to a storage container or containedwithin a filter; nitroxides may be chemically attached to an insolublematrix used in a filter or contained therein in several forms as anadvantageous method of administration, 2) nitroxide covalently linked tohemoglobin that is stabilized by chemical or recombinant cross-linking,3) nitroxide covalently linked to polymerized hemoglobin, in particular,in 2, 4, and 8 molar equivalents of nitroxide, 4) nitroxidecoencapsulated with hemoglobin inside a liposome or intercalated into aliposome membrane, (5) nitroxide covalently bound to conjugatedhemoglobin, (6) nitroxide covalently bound to several forms of albuminin high molar ratios, i.e., between 6 and 95, and (7) nitroxidecovalently bound to immunoglobulins, and any combination of the above ina multicomponent system.

As noted, the above compositions may be used independently or inconnection with low molecular weight, membrane permeable nitroxidesdepending on the application. Moreover, the above compositions may bespecially formulated with other compounds to alter their reactivity orstability in vivo. In particular, cyclodextran and other recognizedstabilizing agents may be used to enhance the stability ofhemoglobin-based solutions. Also, the essential nutrient selenium isknown to generate superoxide and may be used with a polynitroxidemacromolecule to promote the oxidation thereof. These formulations mayalso be used with other known compounds that provide protection fromoxidative stress, which enhance imaging, which increase or decreasesensitivity to radiation, and other known compounds with clinical ordiagnostic utility.

Experimental results are presented below to demonstrate that lowmolecular weight nitroxides may be regenerated from a reduced inactiveform to their active form by interaction with the nitroxide-labelledmacromolecules of this invention. The experimental results andprocedures below show that nitroxides may be attached to biocompatiblemacromolecules, including albumin and stabilized, polymerized,conjugated and encapsulated hemoglobin, for diagnosis therapy, andmeasurement of physiological conditions related to oxidative stress. Theinteraction of nitroxide-labelled hemoglobin and nitroxide-labelledalbumin, both alone and in combination with a low molecular weightnitroxide, with free radicals suggests that other biologicallycompatible macromolecules with a substantial plasma half-life may belabelled with nitroxides and used pursuant to this invention toadvantageously provide resistance to or protection from oxidative stressor toxicity caused by free radical chemical species.

Experimental results are also presented to demonstrate that thecompositions and methods of this invention are anti-hypertensive wheninfused with an HBOC such that the infusion of an HBOC solution isrendered vasoneutral. Radioprotection is demonstrated both with cellcultures and with mice exposed to lethal doses of radiation. EPR imagesof the rat heart are shown which are capable of monitoring the progressof ischemia and reperfusion injury and which demonstrate that, inaddition to image-enhancement, the compositions disclosed herein protectthe ischemic heart from reperfusion injury.

DESCRIPTION OF FIGURES

The file of this application contains at least one photograph/image incolor. Copies of this patent with color figures will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIGS. 1A and 1B show the electron spin resonance spectra of4-amino-TEMPO labelled o-raffinose polymerized hemoglobin recorded on(A) day 1 and (B) day 30 (TEMPO: 2,2,6,6 tetramethylpiperidine-1-oxyl).FIG. 1C is the spectra of the sample in FIG. 1A diluted with equalvolume of unlabelled hemoglobin recorded on day 1. FIG. 1D is the samplein FIG. 1C recorded on day 30.

FIGS. 2A and 2B are, respectively, the electron spin resonance spectrademonstrating covalent attachment of 4-(2-bromoacetamido)-TEMPO toω-aminohexyl-agarose and 4-amino-TEMPO to 1,4-bis(2:3-Epoxypropoxy)butane-activated agarose.

FIGS. 3A and 3B, respectively, are electron spin resonance spectrademonstrating successful covalent attachment of4-(2-Bromoacetamido)-TEMPO and 3-maleimido-PROXYL to3,5-bis-bromosilicyl-bisfumarate (DBBF) cross-linked or diaspirincross-linked human hemoglobin (HBOC).

FIG. 4A is an ESR spectra of 4-(2-bromoacetamido)-TEMPO. FIG. 4B is anESR spectra of 4-(2-bromoacetamido)-TEMPO-labelled HBOC. FIG. 4C is anESR spectra of ¹⁵ ND₁₇ TEMPOL in Lactated Ringer's solution recorded atroom temperature.

FIG. 5 is an ESR spectra of 4-(2-bromoacetamido)-TEMPO-labelled HBOCwith different molar ratios of nitroxides to Hb; FIG. 5A 2:1, FIG. 5B4:1 and FIG. 5C 8:1. The instrument sensitivity were decreasedproportionately from FIG. 5A to FIG. 5B to FIG. 5C to record the spectraso that the center peak (Mo) would be shown to have similar peak height.

FIG. 6 is an ESR spectrum of a mixture of 4-(2-broma-acetamido)-TEMPOlabelled HBOC and ¹⁵ ND₁₇ -TEMPOL wherein the center peak (see downarrow) of the former and the high field peak (see up-arrow) of thelatter were adjusted to similar intensity. This is a superimposition ofESR spectrum from FIG. 4B and FIG. 4C.

FIG. 7 shows the plasma half-life of 4-(2-bromoacetamido)-TEMPO-labelledHBOC in a mouse. FIG. 7A is the ESR spectrum of the nitroxide signalrecorded from the mouse tail approximately 10 minutes after intravenousinfusion of 0.5 ml of the sample shown in FIG. 6. FIG. 7B is the timedependent (scan time 30 minutes) decrease in the center peak (Mo) signalintensity of FIG. 7A recorded at 10 times of the instrument sensitivity.FIG. 7C is a continuation of FIG. 7B at the end of its scan.

FIG. 8 shows the plasma half-life of a mixture of4-(2-bromoacetamido)-TEMPO-labelled HBOC (8g/dl of Hb and 8:1 TEMPO toHb) and ¹⁵ ND₁₇ TEMPOL (0.5 ml in a 32 g. mouse) recorded from the mousetail with a cannula for immediate recording of the infused nitroxides.The ESR spectrum of the sample prior to injection is shown in FIG. 6.FIG. 8A is a series of 5 ESR spectrum recorded at 0.5 minute intervals,the magnetic field strength was increased by 2 Gauss in between eachscan to display the decrease in signal intensity as a function of time.FIG. 8B is the continuation from FIG. 8A of repeated recording of aseries of 6 ESR spectrum at the same time intervals except that themagnetic field strength was decreased by 2 Gauss in between each scan.

FIGS. 9A and 9B, respectively, are electron spin resonance spectrademonstrating 4-amino-TEMPO labelled and o-raffinose cross-linked andpolymerized human hemoglobin and 3-maleimido-PROXYL labelledDBBF-hemoglobin polymerized with glutaldehyde.

FIGS. 10A and 10B, respectively, are electron spin reson ance spectra ofliposome encapsulated human hemoglobin containing (A) 3-DOXYL-cholestane(B) 16-DOXYL-stearic acid. FIG. 10C is the electron spin resonancespectra of both 3-DOXYL-cholestane and 16-DOXYL-stearate.

FIG. 11 is the e lectron spin resonance spectrum of nitroxide-labelledhemoglobin labelled with 4-amino-TEMPO and conjugated with dextran.

FIG. 12 is an embodiment of a filter cartridge that contains a solidmatrix to which a nitroxide is bound and through which ahemoglobin-containing solution may be passed.

FIG. 13 shows the mean arterial pressure (MAP) response in a rat tointravenous infusion of 7.8 g/dl 10% v/v DBBF-Hb alone in Ringer'slactated solution (broken line) and 7. 89/dl 10% v/vDBBF-Hb+polynitroxide albumin (PNA) 5 g/dl+TPL 100 mm 10% v/v (solidline) in conscious rats. The rats were allowed to recover from surgeryand anesthesia for approximately 7 days prior to study.

FIG. 14 is a plot showing time dependence of rat plasma concentrationsof-TPL after intravenous injections. Plasma samples were obtained fromrats described in FIG. 13. TPL concentrations were determined from EPRspin density measurements.

FIGS. 15A, 15B, and 15C, respectively, are electron paramagneticresonance (EPR) spectra of: 15A, TPL (2 mM) in sodium phosphate buffer50 mM, PH 7.6; 15B, TPH (2 mM) in the same buffer; 15C, polynitroxidealbumin (PNA). EPR spectrometer setting conditions as follows. Microwavepower: 8 mW; Receiver gain: 1.00e+03; Modulation amplitude: 0.5 G;Modulation frequency: 100 KHz; Microwave frequency: 9.43 GHz; Sweepwidth: 200 G.

FIG. 16 is a bar graph showing the surviving fraction of Chinese HamsterV79 cells at 12 Gray radiation. The V79 cells were pretreated 10 minutesprior to x-ray irradiation. No radioprotection is observed with TPH orPNA alone (the bar showing 2% survival is same as the control withouttreatment). Increasing radioprotection is shown by the bar correspondingto the sample containing a combination of TPH and PNA (8% survival).

FIG. 17 shows the conversion of TPH to TPL by PNA TPH at fixedconcentration of 25 mM was mixed with increasing concentrations of thePNA in sodium phosphate buffer 50 mM, pH 7.6. The ratio of TPL/TPH wasplotted against 25 mM PNA concentrations. This ratio representsconversion efficiency. TPL concentration was determined by incubating 25mM of TPH and seven different concentrations of PNA at room temperaturefor 30 minutes followed by 10KD membrane centrocon separation for onehour at 5000×g. The high field EPR peak intensities of TPL in thefiltrate were calibrated with TPL standard curve and plotted as shown.

FIG. 18 is a continuous recording of fifteen (15) EPR spectra of themouse tail recorded by manually increasing the field strength byapproximately 1 G in between scans. The scan numbers were marked on thehigh field peak (M-1/2) of the ¹⁵ N TPL. The mouse was previouslyinjected with 0.5 ml of 40 mM ¹⁵ N TPL, which was reduced to TPH with ahalf-life of 2 minutes (results not shown). The second injection of amixture of PNA and ¹⁵ N TPL into the mouse tail vein showed a similarrate of disappearance of ¹⁵ N-TPL (see peaks 1-5 recorded at 30 sec.intervals with a half-life of ˜2 minutes) followed by a equally rapidrecovery of the peak intensity (see peaks 5-7). The peak intensity ofTPL decays with a half-life of 13 minutes (see peaks 7-15, recorded at 1minute intervals). The center (Mo) broad resonance peak (A) ofpolynitroxide albumin (PNA) shown in between the two clusters (M+1/2 andM-1/2) of ¹⁵ N-TPL peaks (TPL) also appeared to decay at a half-life of13 minutes. Thus, the use of ¹⁵ N-TPL clearly demonstrates thespin-transfer from TPH to the ¹⁴ N nitroxides on PNA in vivo.

FIG. 19 shows pharmacokinetics of TPL and TPH in the presence or absenceof PNA in C57 mice. Plasma levels of TPL in arbitrary units asdetermined by monitoring the EPR high field peak intensity were plottedas a function of time field (min.), (□) TPL (2 mM alone) by intravenousadministration, (⋄) TPL 275 mg/kg by intraperitoneal administration and(◯) PNA by intravenous administration followed by TPH 100 mg/kg byintraperitoneal administration.

FIG. 20 shows the 30-day survival study of C57 mice (10 mice per group(N=10) exposed to 10 Gray irradiations after treatment with PNA 0.5ml/mouse by intravenous administration followed by PBS buffer 10 minuteslater (▪), 0.5 ml PBS followed by 200 mg/kg of TPL 10 minutes later byintraperitoneal (ip) administration (), polynitroxide albumin 0.5ml/mouse by intravenous administration followed by 200 mg/kg TPL 10minutes later (♦).

FIG. 21 shows the 30-day survival study of C57 mice exposed to 10 Grayirradiations after treatment with PNA 0.5 ml/mouse by intravenousadministration followed by PBS buffer 10 minutes later (▪), 0.5 ml PBSfollowed by 200 mg/kg of TPL 10 minutes later by intraperitonealadministration (), polynitroxide albumin 0.5 ml/mouse by intravenousadministration followed by 50 mg/kg TPL 10 minutes later (⋄).

FIG. 22A is a 3-D spatial (25×25×25 mm³) EPR image (full view) of therat heart loaded with TPL and polynitroxide albumin. The image wasreconstructed using 144 projections acquired after 2.5 hours ofischemia. FIG. 22B is a cutout view of the same image. Data acquisitionparameters were as follows: spectral window: 7.0 G; spatial window: 25mm; maximum gradient: 49.3 G/cm.

FIG. 23 is an EPR image (25×25 mm²) of the rat heart loaded with TPL andPNA indicating time as a measure of ischemic duration (156 min.),obtained from a 3-D spatial image. Data acquisition parameters were asfollows: acquisition time: 10 min.; spectral window: 7.0 G; spatialwindow: 25 mm; maximum gradient: 49.3 G/cm.

FIG. 24 shows the intensity of ¹⁵ N-Tempol EPR signal in two ischemichearts vs. time of duration of the ischemia. The upper line shows aheart treated with 2 mM TPL+PNA () and the lower line shows a hearttreated 2 mM TPL alone (▪). The solid lines are double-exponentialfittings to the observed intensity data. The half-lives are 0.4 minutes,2.9 minutes (▪), and 3.3 minutes, 30.1 minutes () respectively.

FIG. 25 is a 2-D cross-sectional (25×25 mm²) EPR image of transverseslices of the rat heart loaded with TPL+PNA as a function of ischemicduration with the time indicated digitally (min:sec.) on successiveimages. The images were obtained from 3-D spatial images. Dataacquisition parameters were the same as for FIG. 23.

FIG. 26 shows a measurement of the recovery of coronary flow inuntreated control hearts (◯), treated with TPL 2 mM (), and PNA (4g/dl)+TPL 2 mM (▴). Hearts were subjected to 30 min. of global ischemiafollowed by 45 min. reflow.

FIG. 27 shows the measurement of the recovery of rate pressure product(RPP) in untreated control hearts (◯), treated with TPL 2 mM (), andPNA 4 g/dl+TPL 2 mM (▴). Hearts were subjected to 30 min. of globalischemia followed by 45 min. reflow.

FIG. 28 is a graph of the EPR signal intensity of TEMPOL vs. durationsof ischemia in the rat heart treated with ¹⁵ N-TPL+PNA obtained fromimages shown in FIG. 25. The intensity values were obtained at specificlocations of the corresponding 3-D spatial images as follows: LAD (),Aortic (▪), LV-apex (▾) and tissue (▴).

FIG. 29 shows the mean arterial pressure (MAP) response to intravenousinfusion of 7.8 g/dl 10% v/v DBBF-Hb+PNA 7.5 g/dl+TPL 100 mM 10% v/v(n=4) in conscious rats. The rats were allowed to recover from surgeryand anesthesia for approximately seven days prior to study.

DETAILED DESCRIPTION OF THE INVENTION

Nitroxides are stable free radicals that are shown to have antioxidantcatalytic activities which mimic those of superoxide dismutase (SOD),and which when existing in vivo, can interact with other substances toperform catalase-mimic activity. In the past, nitroxides have been usedin electron spin resonance spectroscopy as "spin labels" for studyingconformational and motional characteristics of biomacromolecules.Nitroxides have also been used to detect reactive free radicalintermediates because their chemical structure provides a stableunpaired electron with well defined hyperfine interactions. In addition,nitroxides have been observed to act as enzyme mimics; certain lowmolecular weight nitroxides have been identified to mimic the activityof superoxide dismutase (SOD). (A. Samuni et. al. J. Biol. Chem.263:17921 (1988)) and catalase (R. J. Mehlhorn et. al., Free Rad. Res.Comm., 17:157 (1992)). Numerous studies also show that nitroxides thatare permeable to cell membranes are capable of short-term protection ofmammalian cells against cytotoxicity from superoxide anion generated byhypoxan-thine/xanthine oxidase and from hydrogen peroxide exposure.

The term "nitroxide" is used herein to describe stable nitroxide freeradicals, their precursors (such as the N-H form), and derivativesthereof including their corresponding hydroxylamine derivative (N-OH)where the oxygen atoms are replaced with a hydroxyl group and exist in ahydrogen halide form. For the purposes of this invention, the chloridesalt form of the hydroxylamine derivatives is generally preferred.

In the nitroxides described here, the unpaired electron is stable inpart because the nitrogen nucleus is attached to two carbon atoms whichare substituted with strong electron donors. With the partial negativecharge on the oxygen of the N-0 bond, the two adjacent carbon atomstogether localize the unpaired electron on the nitrogen nucleus.

Nitroxides generally may have either a heterocyclic or linear structure.The fundamental criterion is a stable free radical. Structurally,nitroxides of the following formula are preferred where R₁ -R₄ areelectron donors and A is the remaining members of a heterocyclic ring.##STR4##

In these heterocyclic structures, "A" represents the remaining carbonatoms of a 5-membered (pyrrolidinyl or PROXYL with one double bond,i.e., pyrroline) or a 6-membered (piperidinyl or TEMPO) heterocyclicstructure and in which one carbon atoms may be substituted with anoxygen atom (oxazolinyl or DOXYL) and certain hydrogen atoms may besubstituted with up to two bromine atoms. In such heterocyclicstructures, stable isotopes may be utilized (e.g., ₁₅ N, deuterium).Substitution at the α carbons should be such that the unpaired electronis maintained substantially in a πp orbital configuration. R₁ through R₄are alkyls (straight and branched chain) or aryl groups but arepreferred to be methyl or ethyl groups. The substituent groups on thealpha carbons in any nitroxide should be strong electron donors toenhance stability, thus methyl (CH₃) groups or ethyl (C₂ H₅) groups arepreferred although other longer carbon chain species could be used. Whenlinked with biocompatible macromolecules pursuant to this invention, thereactivity of the nitroxide is altered due to the microenvironment. Thisreactivity may be tailored by the labelling scheme employed and by thereaction with other compounds, such as selenium, which are known toalter the stability or reactivity of the free radical. In practice,stearic considerations may limit the scope of the nitroxide compoundsthat are practical and economical. The preferred nitroxides used withthis invention include nitroxides having the following structure:##STR5##

As is apparent from the above, most suitable nitroxide compounds may berepresented basically by the structural formula ##STR6## assuming thatthe R group is selected from among the configurations which preserve thestability of the free radical.

A variety of techniques have been described to covalently attach anitroxide to biomacromolecules, including hemoglobin, albumin,immunoglobulins, and liposomes. See e.g., McConnell et. al., Quart. Rev.Biophys. 3: p.91 (1970); Hamilton et. al., "Structural Chemistry andMolecular Biology" A. Rich et. al., eds. W. H. Freeman, San Francisco,p. 115 (1968); Griffith et. al., Acc. Chem. Res. 2: p. 17 (1969); SmithI.C.P. "Biological Applications of Electron Spin Resonance Spectroscopy"Swartz, H. M. et. al., eds., Wiley/Interscience, New York p. 483 (1972).Selected nitroxides have been covalently bound to hemoglobin moleculesfor the purpose of studying cooperative oxygen binding mechanisms ofhemoglobin.

With respect to the macromolecules described here, at least twotechniques for binding the nitroxides to a macromolecule, often known as"labelling strategies" are possible. The significance of specificlabelling lies in the micro-environment in which the nitroxide is boundto the macromolecule and the nitroxide's resulting catalytic activity.Specific labelling at a particular ligand binding site or sites willyield a homogeneous product with a more consistent binding sitemicro-environment and thus a more reliable compound in terms of thecatalytic specificity and activity of the nitroxide.

The term "hemoglobin" is used generally herein to describe oxy-,carboxy-, carbonmonoxy-, and deoxy-hemoglobin except as otherwise notedby the context of the description. The hemoglobin used with thisinvention may be human, recombinant or animal in origin and is obtainedand purified by known techniques. The hemoglobin may be covalently boundto pyridoxal groups of pyridoxa-1-5'-phosphate or ring opened adenosinetriphosphate (o-ATP) by reaction with the aldehyde groups andcross-linked derivatives of hemoglobin. The cross-linked derivatives mayinclude polyfunctional, heterobifunctional and homobifunctionalcross-linking regents such as dialdehyde, polyaldehyde, diepoxide,polyepoxide, activated polycarboxyl and dicarboxyl groups, for example,3,5-bis-bromosilicyl-bisfumarate, and TEMPO succinate or TOPS See (U.S.Pat. No. 4,240,797) cyclodextrans and their anionic (e.c;, sulfate)cross-linked hemoglobin as well as polymerized hemoglobin. Allhemoglobin solutions described herein for use with this invention arephysiologically compatible. The hemoglobin solutions are cell-free toremove pyrogens, endotoxins, and other contaminants.

Preferred compositions using nitroxide in connection with albumininclude:

1) non-specific labelling of albumin with nitroxide (e.g.,4-(2-bromoacetamido)-TEMPO at high nitroxide to albumin ratios;

2) specific labelling of albumin at specific ligand binding sites; and

3) enhanced labelling of albumin by reduction and alkylation ofdisulfide bonds.

As used herein, the term albumin includes human serum albumin, animalalbumin, recombinant albumin, and fragments thereof. The albumin may betemperature or chemical treated to increase the available labellingsites. Additionally, the albumin may exist as a monomer, a dimer, apolymer, or may be enclosed in microspheres. Albumin as disclosed hereinmay also be treated with polyethylene glycol (PEG) by well-knowntechniques to increase its immunocompatibility.

A preferred technique for alleviating oxidative stress by augmenting thebody's antioxidant capabilities is the use of multiple componentnitroxide-based system. A first component is a membrane-permeablenitroxide, such as TEMPOL. By virtue of their charge characteristics andsmall molecular size, low molecular weight unbound nitroxides readilypermeate the cell membrane and enter the intracellular space. A secondcomponent is another nitroxide-including species such as a biocompatiblemacromolecule-labelled with a high molar ratio of nitroxide(polynitroxide), for example, human serum albumin labeled with a 30:1molar ratio of TEMPOL. The use of a multiple component nitroxide systemof this invention helps to alleviate toxicity which could result fromlarge, concentrated, or repeated doses of a low molecular weightmembrane permeable nitroxide. Because the hydroxylamine form of thenitroxide is not active as an antioxidant, and because nitroxidetoxicity at high doses is thought to be primarily due to the antioxidantactivity causing perturbation of the cellular redox state, thehydroxylamine state displays much lower toxicity than the correspondingunreduced nitroxide. Thus, one embodiment of this invention describesthe use of a non-toxic dose of a membrane permeable nitroxide, inconjunction with a macromolecular polynitroxide, to activate a nitroxidein vivo which has been reduced to an inactive form. In similar fashion,the hydroxylamine may be administered as a non-toxic nitroxide precursorwhich is converted to an active antioxidant in vivo. The result is asafe and sustained therapeutic level of a powerful antioxidant in thebody.

With regard to safety in vivo, the levels of nitroxide which may beadministered pursuant to this invention are well tolerated in animalsand are expected to be well tolerated in humans because nitroxides areknown to be relatively safe: For example, the maximum toleratedintraperitoneal dose of TEMPO in mice is 275 mg/kg and the LDQ₅₀ is 341mg/kg. Further, a macromolecule-bound nitroxide will be safer than afree nitroxide in its active form. The nitroxide-labelled macromoleculesof this invention used in combination with free nitroxide will reducethe total quantity of nitroxide that otherwise would have to beadministered to achieve an antioxidant effect. An added advantage ofnitroxide-labelled macromolecules used in antioxidant formulations liesin the ability to achieve high active levels of nitroxides in theiractive anti-oxidant state with improved safety.

Most of the nitroxides studied to date in living organisms have beenrelatively low molecular weight compounds which can easily permeateacross cell membranes into body tissues. The macromolecular-bandnitroxides of this invention may be infused intravenously and may remainconfined to the vascular compartment due to the membrane impermeabilityof the macromolecular species. In such an embodiment, a nitroxide whichis covalently attached to a macromolecule will act to alleviate freeradical toxicity while confining the nitroxide to a location, i.e., thevascular compartment, where the utility is optimized.

When TEMPOL is injected, it diffuses rapidly into the intracellularspace, where it is reduced to the hydroxylamine form in the process ofdetoxifying (oxidizing) free radicals. In the process of scavenging theunpaired electron from a toxic free radical, the nitroxide is reduced toan oxoammonium intermediate. The oxoammonium intermediate can then reactin either of two ways. It can be reoxidized back to a nitroxide byspontaneously donating the free radical-derived electron to somenaturally occurring compound; this pathway may be described asenzyme-mimetic because the net result is that the nitroxide isunchanged. Alternatively, the oxoammonium intermediate can be furtherreduced to a hydroxylamine. The hydroxylamine is not paramagnetic (i.e.,it is silent on EPR spectroscopy) and lacks the antioxidant catalyticactivity of nitroxide. Reaction equilibria in vivo strongly favorreduction to the hydroxylamine. By virtue of its high membranepermeability and inert chemical backbone, the hydroxylamine alsodistributes freely in the intracellular and extracellular spaces, andpersists in the body for a relatively long period of time. However, oncethe nitroxide is reduced to hydroxylamine the antioxidant activity islost.

TEMPOL 4-hydroxyl-2,2,6,6-tetramethyl-piperdine-N-oxyl is rapidlyconsumed in the process of detoxifying free radicals; it is reduced toan oxoammonium intermediate, which can be oxidized back to nitroxide orfurther reduced to a hydroxylamine. Thus, the biotransformation of thenitroxide (in the process of free radical detoxification) yields ahydroxylamine. The hydroxylamine is not paramagnetic (it is silent onesr spectroscopy) and lacks the antioxidant catalytic activity ofnitroxide. The use of TEMPOL alone is not favored therapeuticallybecause it is rapidly converted to the hydroxylamine and may be toxic atthe dosage level needed to achieve a meaningful antioxidant effect.

However, the hydroxylamine is chemically stable and relativelypersistent in the body (the backbone of the nitroxide molecule isrelatively inert) and, in accord with the teachings of this invention,can be chemically converted back to the active form of the nitroxide.This in vivo conversion enables the safe clinical use of nitroxides toprovide a sustained antioxidant activity. As noted above, the unpairednitroxyl electron gives nitroxides other useful properties in additionto the antioxidant activity. In particular, nitroxides in their freeradical form are paramagnetic probes whose EPR signal can reflectmetabolic information, e.g., oxygen tension and information on tissueredox states, in biological systems. Because naturally occurringunpaired electrons are essentially absent in vivo, EPR imaging has thefurther advantage that there is essentially no background noise.Nitroxides also decrease the relaxation times of hydrogen nuclei, andare useful as contrast agents in proton or nuclear magnetic resonanceimaging (MRI). Nitroxides can also act as contrast agents to addmetabolic information to the morphological data already available fromMRI. For example, by substituting various functional groups on thenitroxide, it is possible to manipulate properties including relaxivity,solubility, biodistribution, in vivo stability and tolerance. Contrastenhancement obtained from nitroxide can improve the performance of MRIby differentiating isointense, but histologically dissimilar, tissuesand by facilitating localization and characterization of lesions, suchas blood brain barrier damage, abscesses and tumors.

A macromolecular polynitroxide tends to be distributed in theextracellular space due to its high molecular weight and low membranepermeability and is not readily reduced by the biochemical milieu.However, it has been discovered that the macromolecular polynitroxide iscapable of accepting an electron from the hydroxylamine, causing an invivo conversion back into a nitroxide with active antioxidantcapabilities. This process effectively transfers antioxidant capacityfrom a high-capacity macromolecular storehouse of antioxidant activityoutside the cell, to a high mobility membrane-permeable nitroxide whichmay cross the cell membrane to provide antioxidant activity inside thecell. Once inside the cell, the nitroxide is reduced to thehydroxylamine by oxidizing toxic free radicals, and then cycles out tothe extracellular space, where it is reactivated by the macromolecularpolynitroxide. Moreover, the reactivity of the macromolecularpolynitroxide may be enhanced by adding other compounds such asSelenium.

Therefore, a particularly advantageous nitroxide-containing formulationcan be prepared when a high molar ratio of a nitroxide is bound to amacromolecule (polynitroxide) and allowed to contact a therapeuticallyactive amount of an unbound low molecular weight nitroxide in vivothereby providing a catalytically active polynitroxide macromolecule.The interaction of the therapeutically active nitroxide with acatalytically active nitroxide provides a sustained antioxidant,radioprotectant, imaging agent, etc. in the surrounding tissues. Ineffect, the macromolecular species is a reservoir of antioxidantactivity which can recharge the activity of the low molecular weightspecies which are able to permeate membranes. This symbiotic approachdisclosed herein provides advantageous methods of administration, suchas a topical application of a macromolecular nitroxide combined withoral-administration of a low molecular eight nitroxide to providelocalized, sustained antioxidant activity.

Based on the experimental results and formulation data presented here,the antioxidant, radioprotectant, antihypertensive, and spectroscopicactivity of the nitroxide-containing species of this invention has beenobserved in vitro and in vivo with various formulations. Based on theseresults, the reaction mechanism whereby polynitroxide-labelledmacromolecules and free nitroxides participates in theoxidation/reduction reaction of free radicals is sufficientlydemonstrated that the capability exists to formulate novel HBOCs, andother nitroxide-containing macromolecules to detoxify free radicalswhich will be advantageously used in the diagnosis and treatment of awide variety of physiological conditions.

In addition, this invention describes nitroxide-containing compoundsthat are associated with a container for storage or administration ofpharmaceuticals such as intravenous fluids, topical agents and others.Nitroxide-containing compounds may be added in solid or liquid form tothe interior of a container or may be covalently attached to the innersurface of a container. One advantageous technique for administration isthe addition of nitroxide-containing compounds, with or without freenitroxide, to an in-line filter used in the administration of fluids.For example, a polynitroxide albumin may be incorporated within a filteralong with free nitroxide or are attached to an insoluble matrix housedin a filter to be used with an intravenous fluid administration, such asan existing HBOC to scavenge toxic oxygen-related compounds beforeinfusion into a patient.

The HRCS formulations and nitroxide-labelled macromolecules describedbelow retard the formation of toxic oxygen-related species by causing anitroxide to function as a "superoxide oxidase," an enzyme-like reactionnot known to occur in the red cells. In these HRCS formulations, thenitroxide prevents the accumulation of the undesirable superoxide aniongenerated from the auto-oxidation of hemoglobin (See Equation 1!). Thenitroxide-labelled macromolecules, such as albumin and immunoglobulins,similarly function as antioxidant enzyme mimics whose function remainslocalized in the vascular and interstitial compartments and which mayreact with membrane permeable nitroxides to provide intracellularprotection.

Preferred compositions using nitroxide in connection withimmunoglobulins include a nitroxide-labelled hapten or antigen bound toan immunoglobulin specific for the hapten or antigen.

Furthermore, pursuant to this invention, the beneficial therapeuticeffects of nitroxide compounds can be controlled and sustained. Forexample, the nitroxide 2,2,6,6-tetramethyl-1-oxyl-4-piperidylidenesuccinic acid (TOPS), may be bound to the primary bilirubin binding siteof human serum albumin. In vivo, this binding prolongs plasma half-lifeand slows the diffusion of the nitroxide into the intracellular space,reducing the necessary dosage (and hence reducing potential toxicity)and prolonging biological action. Thus, although nitroxides such as TOPSalone, without a macromolecular polynitroxide, may have utility as anantioxidant agent. Pursuant to this invention, it is possible to selector design carriers which can deliver nitroxides to particular sites inthe body as a means of localizing therapeutic antioxidant activity.

In view of the stable chemical nature of the nitroxides, thecompositions disclosed here can be administered by various routes. Themembrane-permeable nitroxide can be administered parenterally or orally.In the reduced form, hydroxylamine, can act locally in the GI system orbe taken up into the blood. Thus, sustained antioxidant activity can beprovided in all body compartments. The macromolecular polynitroxide canbe administered parenterally where it will remain localized in theextracellular space to reactivate reduced free nitroxide, orally, ortopically/transdermally where it acts to activate circulating, reducednitroxide thereby providing a localized antioxidant effect.

The particular reactivity of a protein-based macromolecularpolynitroxide and a membrane-permeable nitroxide appears to be enhancedby heating of the macromolecule and labeling at primary amino groups inthe polypeptide chain. Heating is known to alter the conformation of themacromolecule, stretching hydrogen bonds between amino and carboxylgroups and causing the macromolecule's quaternary structure to bealtered. Subsequent covalent labeling by nitroxides at the amino groupsmay occur at relatively internal sites on the protein which were exposedas a result of heating. In the resulting nitroxide-labeledmacromolecule, these nitroxide moieties are more protected from reactionwith the solvent. Also, where nitroxides are attached to many aminogroups on the protein, the preponderance of remaining carboxyl groupscreates an acidic microenvironment surrounding the bound nitroxide. Anacidic environment increases the reactivity of the nitroxide by drawingthe unpaired electron in the N-O bond toward the oxygen atom.

In the embodiments of the invention directed to a red cell substitute,the requisite property of the nitroxides is their ability to influencethe course of the superoxide anion cascade in HRCS by mimicking thesuperoxide oxidase, superoxide dismutase, and catalase activitieswithout substantially being consumed in the process.

In the "superoxide oxidase" reaction, the superoxide anion is oxidizedback into molecular oxygen without proceeding to the formation ofhydrogen peroxide. This is accomplished in part by creating a storagecondition wherein the concentration of nitroxide greatly exceeds that ofoxygen. Used in the manner disclosed herein, the nitroxide prevents thecascade of undesirable oxidative reactions that begin with the formationof the superoxide anion. Furthermore, the physiologically compatibleHRCS solutions described here will offer advantages over the existingHBOC solutions because the nitroxide will mimic the enzymatic activityof superoxide dismutase and catalase after the formulations describedherein are infused into a patient.

Although a wide variety of nitroxides may be used with this invention,the nitroxide should be physiologically acceptable at a minimumconcentration required to alleviate oxygen toxicity. In assessing anoperative species, it is noteworthy that the relatively low toxicity ofnitroxides has encouraged their development as contrasting agent in NMRimaging (See U.S. Pat. Nos. 4,834,964; 4,863,717; 5,104,641).

A number of methods for isolating and purifying hemoglobin solutionssuch that they are physiologically compatible are known to those skilledin the art. Typically, purified hemoglobin compositions contain at least99% hemoglobin by weight of total protein, a total phospholipid contentof less than about 3 μg/ml, less than 1 μg/ml of eitherphosphatidylserine or phosphatidylethanolamine and an inactive hemepigment of less than 6%. The purified hemoglobin solutions which areuseful in this invention can be prepared using a variety of conventionaltechniques, including but are not limited to, those disclosed in Cheunget. al., Anal Biochem 137:481-484 (1984), De Venuto et. al., J. Lab.Clin. fled. 89:509-516 (1977), and Lee, et. al., Vith InternationalSymposium on Blood Substitutes, San Diego, Calif. Mar. 17-20 AbstractH51 (1993).

The purified hemoglobin solutions used in this invention should beessentially free of oxygen. Hemoglobin in solution may be deoxygenatedby admixture with a chemical reducing agent which causes the hemoglobinto release oxygen and to be maintained in a substantially deoxygenatedstate. A preferred method for deoxygenating a hemoglobin solution isperformed by exposing a hemoglobin solution to an inert, essentiallyoxygen-free gas, such as nitrogen or carbon monoxide to cause removal ofbound oxygen from the hemoglobin and conversion of the hemoglobin insolution to a form such as deoxy-hemoglobin or carbonmonoxy-hemoglobinthat lacks oxygen. Alternatively, hemoglobin may be exposed to a vacuumor gas through a membrane that is permeable to oxygen yet impermeable tohemoglobin. For example, a hemoglobin solution may be passed through adiffusion cell having a membrane wall along which hemoglobin flows andthrough which oxygen is capable of passing, but hemoglobin is not. Inertgas is circulated along the side of the membrane wall opposite thehemoglobin solution causing the removal of oxygen and the conversion ofthe hemoglobin in solution. to the deoxygenated state. Preferably, thedeoxy-hemoglobin is maintained in an essentially oxygen-free environmentduring nitroxide-labelling, cross-linking, polymerization, orconjugation.

After removal of any bound oxygen, a nitroxide is covalently attached tothe hemoglobin. Normally at least one, and preferably more than one,nitroxide will be covalently attached to a single hemoglobin molecule.The nitroxide may be covalently attached to the hemoglobin at any ofseveral sites on the hemoglobin molecule including:

(a) at one or more of the free sulfhydro (--SH) groups, for example, atthe β-93 site;

(b) at any reactive amino (--NH₂) groups, for example, in the DPG siteat Val-1 of the β-chain and/or lysine-82 of the β-chain and/or lysine-99of the α-chain;

(c) at any non-specific surface amino (--NH₂) or carboxyl (--COOH)group;

A nitroxide may also be bound to any residual aldehyde, epoxy, oractivated carboxyl groups of a divalent--or a multivalent-cross-linkerinvolved in the cross-linking and polymerization of hemoglobin or at anyresidual reactive groups on an agent such as dextran (Dx) orhydroxylethyl-starch (HES) or polyoxyethylene (POE) used to conjugatehemoglobin.

As described in Equation 1!, above, during the storage period, thehemoglobin in an HEOC solution is slowly auto-oxidized by oxygen to formmet-hemoglobin and the superoxide anion. However, during the storage ofthe HRCS that are the subject of this invention, the superoxide anionthus formed will reduce the nitroxide to a hydroxylamine derivative, andthe superoxide anion will be oxidized to form molecular oxygen by thefollowing reaction. ##STR7##

The conversion of superoxide anion to molecular oxygen described inEquation 4! prevents the accumulation of superoxide anion and thesubsequent formation of hydrogen peroxide. This activity, describedherein as a "superoxide oxidase" activity, will be most effective whenthe initial oxygen content in the composition is kept to a minimum, thecomposition is stored in an essentially oxygen free environment and thenitroxide concentration is sufficient to prevent the formation ofsuperoxide anion and hydrogen peroxide. Therefore, storage of the HRCSin an essentially oxygen-free container is preferred.

Container systems that permit a solution to be stored in an oxygen freeenvironment are well known because many non-hemoglobin based intravenoussolutions are sensitive to oxygen. For example, a glass container thatis purged of oxygen during the filling and sealing process may be used.Also, flexible plastic containers are available that may be enclosed inan overwrap to seal against oxygen. Basically, any container thatprevents oxygen from interacting with hemoglobin in solution may beused.

To demonstrate the "superoxide oxidase" activity of a nitroxide, samplesof nitroxide-labelled hemoglobin in solution are kept in an acceleratedoxidative storage condition and the redox state of the nitroxide isstudied over time by electron spin resonance spectroscopy. For example,an o-raffinose polymerized hemoglobin solution that has been labelledwith 4-amino-TEMPO is stored in its oxygenated state in a sealed glasscontainer (FIG. 1A). In such a state, the rate of superoxide anion andmet-hemoglobin formation in solution is sufficiently rapid that theconversion of the nitroxide to its hydroxylamine derivatives may beconveniently monitored (See Equation 4! and compare FIGS. 1A and 1B).Equation 4 represents that the conversion of nitroxide to itsdiamagnetic hydroxyl derivative is coupled to the conversion of thesuperoxide anion back to molecular oxygen. The experimental evidence insupport of such a conversion is shown in FIGS. 1A and 1B. The electronspin resonance spectrum of TEMPO covalently attached to the hemoglobin(FIG. 1A) was converted to its diamagnetic derivatives which result inthe complete disappearance of the resonance peaks after storage of thesample for 30 days at 4° C. (FIG. 1B). The nitroxide is considered tohave performed a "superoxide oxidase"-like activity when it is convertedto its hydroxylamine derivative in the presence of hemoglobin.

The "superoxide dismutase" activity of a nitroxide in an HBOC solutionis demonstrated by showing the reconversion of the hydroxylaminederivative back to a nitroxide (See Equation 5! together with Equation4!). Knowing that under the experimental conditions described in FIGS.1A and 1B the nitroxide is fully converted to hydroxylamine (SeeEquation 4!), the nitroxide may be regenerated by simply providing moresuperoxide anion as shown in Equation 5. To demonstrate this reactionmechanism, the relative concentration of hemoglobin (and thus superoxideanion) to the nitroxide is increased by diluting the sample in FIG. 1Awith an equal volume of unlabelled hemoglobin. A comparison of FIGS. 1Aand 1C shows an approximate 50% reduction of the signal intensity of thenitroxide due to the dilution effect. On the other hand, after 30 daysof cold storage at 4° C., the nitroxide was partially regenerated (SeeFIG. 1D) as predicted by Equation 5!. This observation is consistentwith the reconversion of the hydroxylamine derivative to nitroxidecoupled with the formation of hydrogen peroxide from superoxide anion.##STR8## Summing equations 4! and 5! results in:

    2·O.sub.2.sup.- +2H.sup.+ →O.sub.2 +H.sub.2 O.sub.2

which demonstrates that the nitroxide acts as a low molecular weight,metal-free, SOD mimic in "HBOC" solutions. The detection of electronspin resonance spectrum of the nitroxide (in FIG. 1D) is consistent withthe reaction of superoxide anion with the hydroxylamine (R N--OH)resulting in the formation of nitroxide (R N--O) and hydrogen peroxide(H₂ O₂). Recently, oxoammonium cation has been proposed to be involvedas one intermediate in the nitroxide catalyzed dismutation ofsuperoxide. (Krishna et al., Proc. Nat. Acad. Sci. USA 89 5537-5541(1992)).

The HRCS formulations described herein will alleviate the oxidativestress originating from the generation of the superoxide anion in theexisting HBOC solutions, and upon transfusion, will diminish thedestruction of nitric oxide, the endothelium-derived relaxing factor(EDRF). If the destruction of EDRF is prevented, the problem ofvasoconstriction and systemic hypertension that are observed when theexisting HBOC solutions are infused into a patient will be substantiallyalleviated.

The number of nitroxide molecules per hemoglobin molecule may be in therange of approximately 1-40 and for specific labelling is mostpreferably about 2. When used as a component of a multi-componentsystem, the molar ratio of nitroxide to hemoglobin may be approximately3to 60. For example, a nitroxide such as 3-maleimido-PROXYL iscovalently bound to hemoglobin in solution by first preparing a 100 mMsolution of the nitroxide in ethanol as the carrier solvent. Two (2)molar equivalents of the nitroxide to hemoglobin was added directly withmixing to a DCL-Hb (8 g/dl) in Lactated Ringers. The reaction mixturewas allowed to react at 22° C. with agitation until greater than 90% ofthe nitroxide was covalently linked to the DCL-Hb,usually within onehour. The unreacted nitroxide was then removed with a cross-flowmembrane filtration system having a molecular-weight cut-off of 30,000daltons by washing three (3) times with 10 volumes of Lactated Ringers.The retantate hemoglobin concentration is adjusted to between 7-14 g/dl,sterile filtered, and stored at 4° C. After transfusion, when the HRCSis fully oxygenated, the nitroxide is expected to function as aSOD-mimic and secondly as a catalase-mimic. As an SOD-mimic itdismutates the superoxide anion to hydrogen peroxide (See Equation 2!)and consequently protect against the destruction of nitric oxide in theendothelium to prevent vasoconstriction. As a catalase-mimic it preventshydrogen peroxide toxicity by converting the latter to harmless water(See Equation 3!).

As noted above, nitroxides have been covalently bound to hemoglobin tostudy the cooperative oxygen binding properties of the hemoglobinmolecule itself. However, nitroxides have not been used with stabilized,i.e., cross-linked, or polymerized, encapsulated, or conjugatedhemoglobin solution that are physiologically compatible. The knownchemistry of hemoglobin and nitroxides suggests that it is possible toperform similar nitroxide-labelling of hemoglobin that has beenchemically cross-linked or cross-linked through recombinant techniquesby selecting an available site on the hemoglobin molecule that is notblocked by the particular compound used to stabilize, polymerize, orconjugate the hemoglobin molecule(s). Because certain of the stabilizedand polymerized forms of hemoglobin described below are currentlyinvolved in clinical trials, the attachment of nitroxides to thesestabilized and polymerized hemoglobin-based oxygen carriers is describedbelow to demonstrate that the oxygen detoxification function of thisinvention is applicable to the existing hemoglobin solutions.

The nitroxide-labelling technology demonstrated here in the example ofnitroxide-HBOC is readily applied to the production of othernitroxide-labelled macromolecules with useful antioxidant andenzyme-mimetic activities, for example nitroxide-labelled serum albuminand nitroxide-labelled immunoglobulin. Forms of serum albumin which canreadily be labelled by nitroxide by this technology are monomeric(normal) albumin, and albumin homodimers, oligomers, and aggregates(microspheres) and polypeptide fragments of each.

Due to the differences in application, the formulations described hereinmay be used together or in isolation. For example, in the therapy anddiagnoses of cardiac reperfusion injury, it may be desirable to takeadvantage of several aspects of this invention, i.e., oxygen delivery,systemic protection from oxidative stress, localized protection fromreperfusion injury, and enhanced imaging. In such a case, a combinationof the formulations described herein could be used such as an existingHBOC, nitroxide-labelled albumin and a low molecular weight nitroxidewhich could be administered simultaneously or in sequence, depending onthe therapeutic or diagnostic goal.

With respect to selecting a particular formulation and method ofadministration pursuant to this invention, the formulation and method ofadministration are dictated by the particular application. The selectionof a nitroxide-based compound capable of accepting an electron from alow molecular weight membrane permeable species for a particularapplication may be made to complement, several available methods ofadministration and preferred formulations may be selected based on thesite specific protection desired for the particular application. Theavoidance of oxidative stress from infusion of a hemoglobulin-basedoxygen carrier as described abov is a prime example of selectingformulations and methods of administration pursuant to this invention toprovide specific protection from free radical toxicity to avoid thetoxic side effects of HBOC infusion. Where site specific protection oractivity is desired in the skin or dermal layers a preferred compound isTOPS because it is relatively small and membrane permeable. Wherespecific protection or activity is desired in the gastrointestinaltract, a polynitroxide dextran is preferred because such a compound isless susceptible to enzymatic digestion while in the gastrointestinaltract. In such an application, oral or rectal administration ispreferred. Where specific protection or activity is sought for theintravenous or intravascular regions, such as the cardiovascular system,a polynitroxide albumin is preferred because albumin is a major plasmaprotein, is well-tolerated, easy to administer, and exhibits an extendedplasma half-life. Such application may include a hemoglobin-based oxygencarrier or polynitroxide derivative thereof. The same rationale appliesfor intraperitoneal or intradermal administration. If specificprotection in the lungs is desired, an aerosol form of polynitroxidealbumin is preferred to enable coating of the pulmonary airways. As willbe apparent to those skilled in the art, these preferred formulationsmay be altered depending on the particular application.

As noted, the above formulations are preferred embodiments of theelectron accepting compound. With respect to the membrane permeableelectron donating compound, the selection of a preferred compound alsodepends on the application and method of administration. The formulationand method of administration should achieve a systemic or tissuespecific distribution commensurate in scope with the extent of the freeradical species generated by, or coincident with, the physiologiccondition of interest or the region to be imaged or treated. Forexample, in sepsis, a large scale collapse of the circulatory system isobserved which accompanies a systemic increase in free radicalgeneration. Similarly, whole body irradiation results in free radicalgeneration throughout the body. In such situations, a small molecularweight membrane permeable electron-donating nitroxide such as TEMPOL maybe administered orally or intravenously in a quantity to insure systemicdistribution. Accordingly, such administration should be accompanied byadministration of an electron acceptor which is also widely distributedsuch as an intravenous administration of polynitroxide albumin. Wherethe free radical generation is more localized, it is preferred toprovide the localized protection or activity by selective application ofthe electron accepting species because the membrane permeable speciestends to become systemically distributed upon administration, although atopical administration of the membrane permeable species to the skin mayyield a more localized effect.

Based on the disclosure herein, those skilled in the art can selectformulations and methods of administration to most effectively meet thedemands of the particular application where this invention is to beused. For example, to achieve image enhancement of the gastrointestinalsystem, one may select an oral administration of polynitroxide dextranas the electron acceptor and an oral and/or intravenous administrationof membrane permeable TEMPOL. As a further example, to treat psoriasis,a localized skin condition where free radical generation is manifested,one may select a topical application of TOPS as the electron acceptorand a topic application of membrane permeable TEMPOL. By any similarrationale, site specific protection or activity can be provided usingthe formulation of this invention for other disease states, ordiagnostic or therapeutic state where free radical species are present.Therefore, the following examples disclose a detailed description ofseveral formulations which can be used in any combination depending onthe application to which this invention is applied.

EXAMPLE ONE Containers and Filters Containing Nitroxides andNitroxide-Labelled Compounds

It is possible to provide the oxygen-detoxification function of thisinvention to existing intravenous solutions, such as the HBOC solutions,without chemically modifying the existing formulations. By including apolynitroxide macromolecule, which may be used in connection with a freenitroxide, or by covalently attaching nitroxides to a surface inside thevessel in which the HBOC is stored, the adverse physiological effectscaused by oxygen toxicity that are observed with the existingformulations will be alleviated.

The container used with the hemoglobin-containing solutions that are thesubject of this invention should be physiologically compatible havingsimilar characteristics as any container to be used with intravenousfluids. Typically, glass or a biocompatible plastic is suitable. For theembodiments of the invention where an intravenous solution is placed ina container for any length of time, the container should be oxygen freeand capable of being sealed to exclude oxygen. With a glass container, atraditional stopper and closure means is sufficient. However, some ofthe flexible plastic containers currently available are oxygenpermeable. In this case, a foil overwrap or other sealing mechanism maybe used to prevent oxygen from contacting the solution.

To apply a nitroxide to an inner surface of a container, a non-leachinglayer of a nitroxide polymer or a nitroxide-doped copolymer is coateddirectly on the inner surface. Nitroxide-containing polymers can becreated by a number of techniques based on generally known principles ofpolymerization as long as the stability of the free radical ismaintained in the polymerization process.

Also, the interior surface of an HBOC container may be modified tocontain a coating layer of a substance that can bind a nitroxide, suchas hydrophilic hydrazide groups which will react with the ketone or thealdehyde group of a nitroxide to form stable hydrazone derivatives. Thecoating reaction is straight forward. For example, the nitroxide (100mM) in acetate buffer at pH 5.0 is added to a hydrazide activatedplastic container to facilitate the formation of a hydrazone linkage.

Once the container is prepared, a physiologically compatible solution isadded. This solution may be a stabilized and purified HBOC or the HRCSdisclosed herein, and could also include any intravenous colloid orcrystalloid solution that is desirable to co-infuse with hemoglobin. Thesolution is then maintained in an essentially oxygen-free environment.

In addition to treating a surface inside a container, a filter-typecartridge, with a luer lock inlet and outlet, containing a gel or solidmatrix upon which a nitroxide is immobilized may be used to removereactive oxygen-derived reactive species while the hemoglobin solutionpasses through the cartridge. For such an administration technique, apolynitroxide macromolecule may be added into the housing of the filterthrough which a solution passes for direct infusion into a patient toreact with the solution before infusion. A low molecular weightnitroxide may also be included. In these applications, nitroxide mayalso be bound to a soft--or hard-gel matrix, thereby functioningessentially as a sterile in-line filter, prior to infusion. A variety ofmethods to attach small ligands, such as nitroxide, to a solid matrixare well known in the art of affinity chromatography, as are thetechniques to chemically modify glass and plastic surfaces. Severaltypes of matrices that are compatible with sterile solutions are knownincluding agarose gel, polysulfone-based material, latex, and others.

In the filter cartridge approach, the solid matrix is covalently linkedwith a nitroxide and contained in a filter housing or other suchapparatus such that a solution, such as hemoglobin can flow through theapparatus and be brought into contact with a nitroxide while beinginfused into a patient. A practical approach is to use a commonlyavailable activated agarose gel as the matrix and contain the gel in asterile luer lock cartridge. The cartridge is then simply inserted inthe fluid administration line during the transfusion of a solutioncontaining hemoglobin. In practice, the structure that comprises thefilter housing in which the nitroxide and through which hemoglobin ispassed can be provided by a variety of known structures. See e.g., U.S.Pat. No. 5,149,425. Referring now to FIG. 12, housing 1 contains anitroxide-labelled agarose gel. For example, a 4-bromoacetamido-TEMPOlabelled ω-aminohexyl-agarose (See FIG. 2A) a1,4-bis(2,3-epoxypropoxy)butane agarose coupled with 4-amino-TEMPO (SeeFIG. 2B). Other compounds (not shown) may be included with the filterhousing.

During the transfusion, the intravenous transfusion line containing thesolution would be connected to the luer inlet 2 allowed to enter thehousing 1 wherein the hemoglobin solution would encounter thenitroxide-containing compounds contained within the housing or bound tothe matrix 4, in this process, the nitroxide-containing compositionswill be infused and may react to remove the toxic oxygen-related speciesfrom solution. The hemoglobin solution would then pass out of thecartridge through the luer outlet 3 and would be directly transfusedinto a patient. The electron resonance spectrum of 4-amino-TEMPOlabelled epoxy-agarose is shown in FIG. 2A. Alternatively, anω-aminohexyl-agarose may be reacted with 4-(2-bromoacetamido)-TEMPO toform TEMPO labelled agarose. The electron spin resonance spectrum isshown in FIG. 2B. An alternative would be to couple the 4-carboxyl-TEMPOto the amino-agarose with carbodiimide via a carboamide linkage.Conversely, the 4-amino-TEMPO is readily coupled to the carboxyl groupon an agarose gel using carbodiimide, for example,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

The cartridge labelled with 4-amino-TEMPO prepared by circulating a 100mM 4-amino-TEMPO (Sigma Chem. Co.) in a Lactated Ringers solutionthrough an aldehyde AvidChrom Cartridge (Unisyn Tech. Inc.) at roomtemperature for one hour followed by the reduction with sodiumcyanoborohydride for six (6) hours. The interior of the cartridgehousing is thoroughly washed with Lactated Ringers.

The cartridge labelled with 3-amino-PROXYL may be similarly prepared bysubstituting 4-amino-TEMPO with 3-amino-PROXYL according to theprocedure described above.

EXAMPLE TWO Nitroxide-Labelled Stabilized Hemoglobin

To prevent dissociation of hemoglobin into its constituent subunits,hemoglobin is intramolecularly stabilized by chemical or recombinantcross-linking of its subunits. "Stabilized" hemoglobin is referredherein to describe hemoglobin monomers that are stabilized by chemicalor recombinant cross-linking and also to describe dimers, trimers, andlarger oligomers whose constituent hemoglobin molecules are stabilizedby cross-linking with cyclodextrans and their sulfated derivatives.

A preferred technique for attaching nitroxide to stabilized hemoglobinis by the covalent attachment of the nitroxide to the β-93 sulfhydrylgroups of the two β-chains of stabilized hemoglobin. Although specificlabelling at the β-93 site has been demonstrated on native humanhemoglobin for conformational studies (See review by McConnell et. al.,Quart. Rev. Biophys. 3:91 (1970)), such a specific labelling ofcross-linked hemoglobin has not been reported. As noted above, severaltypes of hemoglobin-based oxygen carriers have been developed that arestabilized through chemical cross-linking with DBBF, diaspirincross-linked hemoglobin and hemoglobin that is stabilized andoligomerized with o-raffinose.

The ring opened sugars described in my U.S. Pat. No. 4,857,636 yieldpolyvalent aldehydes derived from disaccharides, oligosaccharides, or,preferably, trisaccharides such as o-raffinose. These compounds functionboth to provide intramolecular stabilization (cross-linking) andintermolecular polymerization. By controlling the reaction disclosed inmy earlier patent, the polyvalent aldehydes may be used to produce"stabilized" hemoglobin as defined above without polymerization. Inanother case, a nitroxide may be covalently bound to the stabilizedhemoglobin or the polymerized hemoglobin. Therefore, thehemoglobin-based solutions that are stabilized using the polyvalentaldehydes are considered in the present embodiment as a "stabilized"hemoglobin and in the subsequent embodiment as a polymerized hemoglobin.

To demonstrate, that the β-93 site of the chemically modified hemoglobinhas not been rendered sterically inaccessible for nitroxide attachment,results are presented to confirm that a nitroxide may be covalentlybound to the β-93 site of DBBF-Hb.

In this embodiment, DBBF-Hb is reacted with two types of nitroxides(TEMPO and PROXYL) which contain two types of sulfhydro group specificfunctional groups and have the following structural formula: ##STR9##

DBBF-Hb is prepared by cross-linking purified deoxygenated hemoglobin insolution with bis(3,5 dibromosalicyl)fumarate by known techniques, andthe resulting product is purified by column chromatography. The covalentattachment of 3-maleimido (2,2,5,5 -tetramethyl pyrrolidine-N-Oxyl)3-maleimido-PROXYL! is accomplished by adding 2 molar equivalents ofthis nitroxide using methanol as the carrier solvent at a concentrationof approximately 100 mM of 3-maleimido-PROXYL to 1 ml of DBBF-Hb at aconcentration of approximately 8 g/dl in Lactate Ringers. The DBBF-Hb isallowed to react at 22-23° C. for approximately 30 minutes with mixing.The extent of cross-linking is estimated from the percent disappearanceof the electron spin resonance signal intensity of the unreactednitroxide. To remove the unreacted nitroxide, the reaction mixture waswashed three (3) times with a 10 volume excess of Lactated Ringers usinga Filtron stire cell with a 30 kilodalton cut-off nominal molecularweight limits (NMWL) polyethylene sulfone (PES) membrane (FiltronTechnology Co.). The electron spin resonance measurements of thenitroxide-labelled hemoglobin was recorded with a Bruker ESRspectrometer. FIG. 3A shows the electron spin resonance spectra of4-(2-bromoacetamido)-TEMPO labelled DBBF-Hb. The electron spin resonancespectrum of DBBF-Hb that is similarly labelled with 3-maleimido-PROXYLis shown in FIG. 3B.

In this embodiment, the nitroxide is covalently linked to the lonesulfhydro group on the two β-globin chains of hemoglobin. Thus, thenitroxide to hemoglobin-bound oxygen ratio is approximately 200 to 1 at99.00% deoxy-hemoglobin because there are two nitroxides attached to thetwo β-globin chains of the hemoglobin. After transfusion, however, thedeoxygenated HRCS picks up oxygen in the lung and the nitroxide tohemoglobin-bound oxygen ratio becomes approximately 1 to 2 at 100%oxygenation because there are four oxygen molecules bound to the fourglobin chains of the hemoglobin with the two nitroxides remaining on theβ-globin chains.

Using a hemoglobin-to-nitroxide ratio of 1:2, greater than 90% of thenitroxide is covalently attached to the DBBF-Hb. DBBF-Hb may also becovalently labelled with a spacer group (e.g., an extra methyl group)between the maleimido and PROXYL moieties (i.e.,3-maleimidomethyl-PROXYL) which would exhibit a resonance spectrumsimilar to that of FIG. 3B. It is noteworthy that other nitroxides maybe covalently attached to specific amino-groups in the DPG binding site(e.g., β-Val-1 β-Lys-82 and ∝-Lys-99) or may be attached to theremaining 40-plus surface lysine ε-amino groups on hemoglobin.Isothiocyanate derivatives of the TEMPO and PROXYL nitroxides are alsoreactive with the amino group. For example, 4-isothiocyanate-TEMPO maybe added to hemoglobin at a molar ratio of approximately 10:1. Resonancespectrum (not shown) of hemoglobin labelled with this nitroxide at othersites is similar to that shown in FIG. 3A.

The ability to attach nitroxides at several sites of DBBF-Hb suggeststhat recombinant hemoglobin that is stabilized with alpha-globin dimers(D. Looker et.al. NATURE 356:258 (1992)) may be similarly labelled witha nitroxide. It is also possible to prepare a DBBF analogue of anitroxide-labelled cross-linking agent such as a TEMPO labelledsuccinate (See U.S. Pat. No. 4,240,797).

FIG. 4 is ESR spectra of (A) 2-(bromoacetamido)-TEMPO, (B)2-(bromoacetamido)-TEMPO-labelled HBOC and (C) ¹⁵ ND₁₇ TEMPOL (TEMPOL:4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) in Lactated Ringer'ssolution recorded at room temperature. The difference in FIGS. 4A and 4Brepresent the difference in the mobility of a small molecular weightnitroxide to that of a nitroxide covalently attached to a macromoleculesuch as hemoglobin. FIG. 4C shows that a stable isotope nitrogen ¹⁵ Nwith a nuclear spin of 1/2 yields two resonance peaks and thatnatural-isotopic ¹⁴ N with a nuclear spin of 1 yields three resonancepeaks (compare (4A to 4C). In the set of experiments described here theseparation of these resonance peaks is used to demonstrate theenzyme-mimic and in vivo and in vitro oxidation/reduction reactions ofsmall and macromolecular weight nitroxides.

Nitroxide-labelled HBOC with different molar ratios of nitroxide tohemoglobin are prepared as follows. 2, 4, and 8 molar equivalents of4-(2-bromoacetamido)-TEMPO, were added as solid powder directly intothree separate 15 ml Vacutainers in a clean hood. After replacing therubber septum, 4-(2-bromoacetamido)-TEMPO was subsequently dissolved in200 ul chloroform. The Vacutainers were then connected to high vacuum (5mm Hg) via a 27 gauge needle through the rubber septum and thechloroform was removed leaving a thin film of 4-(2-bromoacetamido)-TEMPOcoating the lower half of the Vacutainer. After introducing theappropriate amount of HBOC via sterile transfer through the rubberseptum, the solutions were allowed to react at room temperature withintermittent vortex mixing at approximately 5 minute intervals for 1/2hour (not all solids were dissolved in the 4 and 8 molar ratios ofnitroxide to hemoglobin), the Vacutainers were then left at 4 degrees C.in a refrigerator over night. Vortex mixing at room temperature wasresumed the next morning for another 1/2 hour until all solids of4-(2-bromoacetamido)-TEMPO had visually disappeared from the surface ofthe Vacutainer.

The reaction mixtures and the control, were then transferred to asterile dialyzing tube and dialyzed against Lactated Ringers until nounlabelled free 4-(2-bromoacet-amido)-TEMPO electron spin resonance(ESR) signals could be detected. The ESR spectra of4-(2-bromoacetamido)-TEMPO-labelled HBOC at 2, 4, and 8 molarequivalents 4-(2-bromo-acetamido)-TEMPO to Hb are shown in FIGS. 5A-5Crespectively. At 2 molar equivalents of 4-(2-bromoacetamido)-TEMPO tohemoglobin, the ESR spectra are essentially the same with or withoutdialysis indicating the covalent labeling is quantitative. The two SH--groups on the beta globulin chains appear to be the site of covalentattachment in the case of HBOC (this can be confirmed by selectiveblocking of 4-(2-bromoacetamido)-TEMPO labeling with N-ethyl-maleimideor globulin chain analysis by reverse phase HPLC). It is noteworthy thatthe ESR signal intensity (peak Mo) ratios for 2, 4, and 8 are inapproximately the same ratio as the spectra were recorded atproportionately decreasing instrument sensitivity.

Furthermore, it is expected that more 4-(2-bromoacetamido)-TEMPO couldbe attached to Hb at even higher molar ratios, for example asradiation-protective agents in vivo.

The preferred molar of nitroxide to hemoglobin in the blood substituteformulations is 8:1 as described below.

Referring to FIG. 6, an ESR spectrum of a mixture of4-(2-bromoacetamide)-TEMPO-labelled HBOC and ¹⁵ ND₁₇ -TEMPOL wherein thecenter peak of the 4-(2-bromoacetamido)-TEMPO (indicated by down arrow)and the high field peak of ¹⁵ ND₁₇ -TEMPOL (indicated by the up-arrow)were adjusted to similar intensity.

The separation of the resonance peaks permits the simultaneousmonitoring of free radical or enzyme mimic activities involving thesmall molecular weight nitroxide (TEMPOL) and its macromolecularconjugate in both in vitro and in vivo (murine) reactions. For example,the in vivo plasma half-life of the two nitroxides was compared byreferring to the unique spectral characteristics of the differentnitroxides. Specifically, the in vivo ESR studies of hemoglobin-basedsolutions, on the mouse were performed using a nitroxide to hemoglobinratio of 8:1 (see FIG. 5C) to take advantage of its high ESR signalintensity. First, the approximate plasma half-life of a small molecularweight nitroxide (¹⁵ ND₁₇ -TEMPOL see FIG. 4C) and a large molecularweight 4-(2-bromoacetamido)-TEMPOL-labelled HBOC (see FIG. 4B) aredetermined by preparing a mixture of the two and adjusting the ESRsignal intensity to be approximately the same (see FIG. 6). 0.5 ml ofthe mixture was injected under anesthesia into a distended mouse tailvein under a heat lamp. The mouse tail was inserted into an ESR cavityand the spectrum was recorded within 10 minutes after injection (seeFIG. 7A).

Referring to FIGS. 7A, 7B, and 7C the ¹⁵ ND₁₇ -TEMPOL signal could notbe detected, however, the 4-(2-bromoacet-amido)-TEMPO-labelled HBOC wasclearly resolved (see FIG. 7B and 7C for plasma half-life studies where7C is a continuation of 7B). Since the vasoconstrictive effect of HBOCis reported to be fully developed during the first 5-15 minutes of bolusinjection of an HBOC in rats, the participation of thenitroxide-labelled HBOC in free radical redox-reactions immediatelyafter transfusion in a mouse was measured. The tail vein of female CH3mouse was cannulated under anesthesia with 80% nitrous oxide, 20%oxygen, and 3% isofluorane. Under a heat lamp the mouse tail vein becamevisibly distended, a cannula consisting of a 30 gauge hypodermic needleattached to a one foot length of polyethylene tubing was inserted intothe tail vein and held in place by cyanoacrylate glue. For in vivo ESRmeasurements, the cannulated mouse was transferred under anesthesia to a50 ml conical centrifuge tube modified to allow the tail to protrudefrom the conical end and to allow a continuous flow of anesthetic gasfrom the opening end of the tube. The tail was inserted into a plastictube which was then fitted into a TE 102 cavity. The cannula was flushedperiodically with heparin (100 unit/ml) to ensure patency. The cannulawas near the root of the tail and was kept outside of the ESR cavity sothat a pure signal from the tail could be measured immediately afterbolus injection. 0.5 ml of samples (see FIG. 8) were injected via thecannula and the spectrometer was set for a repeat scanning mode at 1/2min. intervals (see FIGS. 8A and 8B). In FIG. 8A the magnetic field wasincreased by two Gauss, and in FIG. 8B the magnetic field was decreasedby two Gauss, to superimpose the resonance spectra. The ¹⁵ ND₁₇ -TEMPOLsignal disappeared within 2.5 minutes after injection. During the sametime period the 4-(2-bromoacetamido)-TEMPOL-labelled HBOC also decreasedat a similar rate.

However, the nitroxide-HBOC signal were shown to be stable in plasma(FIG. 8B). Therefore, FIG. 8B together with results from FIG. 7 showthat the nitroxide-labelled to macromolecules such as HBOC hasconsiderably longer plasma half-life as compared to small molecularweight nitroxide (e.g., ¹⁵ ND₁₇ -TEMPOL).

The observed nature of the free radical reaction involves two pathways:

1. the rapid phase appears to involve the free radical (e.g. superoxide)oxidation of the nitroxide to its oxoammonium cation intermediatefollowed by the reduction of the oxoammonium cation to its stablehydroxylamine derivative of the nitroxide. Such reduction involves theparticipation of either one or two reducing equivalents (e.g. NADH)present in the vascular compartment. The reduction of nitroxide to itshydroxylamine would lead to a rapid reduction in ESR signal intensity,in the case of 8:1 molar ratio of 4-(2-bromoacetamido)-TEMPO-labelledHBOC represents approximately 25% of the 4-(2-bromoaceta-mido)-TEMPO onthe HBOC. This phase involves both small molecule and macromolecularnitroxide.

2. the slow phase appears (see FIG. 8B) to represent the antioxidantenzyme-mimic activities of the remaining 75% of4-(2-bromoacetamido)-TEMPO on the HBOC in accordance with the reactionmechanism wherein the nitroxide is involved in the cyclic-free radicalreactions for example the SOD-mimic reaction. Where the nitroxide freeradical is essentially unconsumed as a SOD-mimic, the slow rate ofdecrease of the ESR signal intensity can be attributable primarily tothe reaction mechanism described above and secondarily to the decreasein HBOC concentration as it is slowly eliminated from the vascularcompartment as a function of its plasma half-life.

This result demonstrates the utility of polynitroxide macromolecule, inthis example TEMPO-labelled HBOC, in detoxifying free radicals in vivo.This utility is defined in terms of providing short term (in minutes)scavenging of free radicals and persistent (in hours) protection againstoxidant reactions by nitroxides acting as enzyme mimics in vivo. In thisand each of the examples related to hemoglobin-containing solutionshould be understood that unbound, low molecular weight nitroxide may beadded to the formulation to increase the concentration of activenitroxide across the vascular membrane, into the interstitial space, andthe surrounding cellular environment. The results presented here therebydistinguish the effect of a simple addition of a low molecular weightnitroxide to a pharmaceutical composition from the polynitroxidemacromolecules of this invention. The particular advantages of amulti-component system of this invention utilizing a polynitroxidemacromolecule together with molecular weight nitroxides is highlightedbelow.

Based on the analysis of the spectra in FIG. 8, the oxidation/reduction(redox) cycling reactions involve approximately 73% of4-(2-bromoacetamido)-TEMPO-labelled HBOC remaining in its free radicalstate. This indicates that TEMPOL participates in in vivoredox-reactions within the confines of the vascular space.

To study the vasoconstrictive effects of hemoglobin-based solutions,solutions of modified human hemoglobin are tested for their effects inthe intact rat. Humane procedures are always used where any researchanimals are used.

At 7 days prior to the study, male Sprague-Dawley rats are anesthetizedwith ketamine (40 mg/kg i.m.) and acetylpromazine (0.75 mg), or withpentobarbital sodium (20 mg/kg i.p.). Medical grade Tygon microbore(0.05 in ID, 0.03 in OD) is inserted into the femoral artery and veins.Cannulas are exteriorized and filled with heparinized dextrose, andsealed with stainless steel pins. After a 2-3 day recovery period,conscious animals will are in plastic restraining cages. Seven (7) daysrecovery from surgical procedure are needed to ensure healing ofincisions before exchange transfusion. Because the surgery may causeminor bleeding, it is important to permit recovery so that minorbleeding related to surgery is not confused with a side effect of bloodreplacement. 50% exchange transfusions are carried out using an infusionpump to simultaneously infuse and withdraw the test solution and blood,respectively, from two syringes. The volume of blood removed (12-15 mlbased on total blood volume) is replaced with test solution overapproximately 20-30 minutes. The end point is the reduction of thehematocrit to half its initial value. The arterial blood pressure ismonitored and recorded continuously for 5 hours after the exchangetransfusion using a pressure transducer connected to a chart recorder.Mean arterial pressure is calculated as 1/3 of the pulse pressure. Heartrate is determined from the blood pressure trace.

EXAMPLE THREE Nitroxide-Labelled Polymers of Stabilized Hemoglobin

While it is possible to produce dimers of stabilized hemoglobin fromcross-linked monomers, it is also possible to produce hemoglobinpolymers from stabilized or native hemoglobin. Solutions of hemoglobinpolymers contain a mixture of monomers, dimers, trimers, tetramers, andother oligomers. Solutions containing polymerized hemoglobins used as anHBOC generally have longer plasma circulation times and higher oxygencarrying capacities as compared to stabilized monomeric hemoglobin. Suchpolymerized hemoglobin may be prepared by a number of pathways usingseveral different polymerizing agents. (See, U.S. Pat. Nos. 4,001,200,4,857,636, and 4,826,811). The preferred method of introducing anitroxide to a solution of polymerized hemoglobin is again by covalentlyattaching a nitroxide to the β-93 sulfhydryl groups of the two β-globinchains of hemoglobin. These sulfhydryl groups are not known to beinvolved in the stabilization or polymerization processes. Consequently,the nitroxide is preferably covalently attached to hemoglobin before thestabilization and polymerization of the hemoglobin monomers.

For example, nitroxide is covalently attached to DBBF-Hb according tothe procedure described in the second embodiment above, followed bypolymerization with glutaldehyde according to the procedure described inSehgal et. al. U.S. Pat. No. 4,826,811. FIG. 4B is an electron spinresonance spectra of the DBBF-Hb labelled with 3-maleimido-PROXYL andpolymerized with glutaldehyde. Similarly, DBBF-Hb that is polymerizedwith glutaldehyde may be labelled with 4-(2-bromo-acetamido)-TEMPO bythe same method.

Using a similar approach, a polymerized hemoglobin intermediate, such asa glutaldehyde-polymerized, an o-raffinose-polymerized, or ano-cyclodextran-polymerized, hemoglobin intermediate that containsunreacted aldehyde groups, may be used for covalent attachment of either4-amino-TEMPO or 3-amino-PROXYL via reductive amination to yield anitroxide-labelled hemoglobin polymer. With reductive amination, thesequence and timing of the reaction are important. The 4-amino-TEMPO isadded to glutaldehyde-polymerized hemoglobin after completion ofpolymerization, but prior to the reduction reaction that results incovalent attachment of the nitroxide to the polymerized hemoglobin.Likewise, the nitroxide-labelling of a o-raffinose polymerizedhemoglobin may be accomplished by the addition of either 4-amino-TEMPOor 3-amino-PROXYL prior to reductive amination. For example,4-amino-TEMPO labelled o-raffinose polymerized hemoglobin is preparedaccording to the procedure described in my U.S. Pat. No. 4,857,636except that 6 molar equivalents of 4-amino-TEMPO is added after thecompletion of the polymerization and prior to the reduction with 20molar excess of borane dimethylamine complex. As described therein,hemoglobin may be cross-linked and polymerized using polyvalentaldehydes derived from disaccharides or ring-opened sugars including,oligosaccharides, or preferably, trisaccharides such as o-raffinose.Likewise, monosaccharides may be used to stabilize and polymerizehemoglobin although the higher molecular weight sugars are preferred.The resonance spectrum of a dialyzed and washed o-raffinose polymerizedhemoglobin labelled with 4-amino-TEMPO was shown in FIG. 9A.

To increase the yield of hemoglobin oligomers (Hb_(n) where n=2-4) ofthe polymerized hemoglobin, it is desirable to increase the valance ofthe polyaldehyde of the cross-linker, with the use of α-cyclodextran,β-cyclodextran, and γ-cyclodextran, as well as their sulfate derivativeswhich represents 6-, 7-, and 8-cyclized glucose molecules, the ringopened α-cyclodextran, β-cyclodextran, and γ-cyclodextran have 12, 14,and 16 reactive aldehyde groups respectively. These ring-openedcross-linkers can be used to cross-link and polymerize hemoglobin toproduce polymerized hemoglobin which is rich in oligomers. The unreactedaldehyde, as described above, may be utilized to covalently attached toan amino-nitroxide, for example, 4-amino-TEMPO or 3-amino-PROXYL.

Furthermore, the ring-opened sulfate derivatives, for example, thesulfated α-cyclodextran will be an effective cross-linker for twoadditional reasons: (1) the sulfate groups will mimic the activity ofDPG in lowering the oxygen affinity of the cross-linked hemoglobin, thusimproving oxygen transport properties, and (2) the sulfate groups willserve as affinity labels which will complex multiple (e.g., n=2-4)hemoglobins to initially form a "cluster." Once the "cluster" complex isformed, the aldehyde groups on the cyclodextran will be brought to closeproximity with the NH2 groups within the DPG binding sites, thuspromoting the covalent intra-subunit and inter-molecular cross-linkingof hemoglobin resulting in an increased yield of hemoglobin oligomers.In addition to antioxidant enzyme-mimic activities, the ring-openedcyclodextran polymerized and nitroxide-labelled hemoglobin will alsohave improved yield and composition as compared to o-raffinose andglutaldehyde polymerized hemoglobin.

EXAMPLE FOUR Nitroxide-Labelled Liposome-Encapsulated Hemoglobin

Liposomes are particles which are formed from the aggregation ofamphophilic molecules to form a bilayer structure in a hollow sphericalshape with the polar sides facing an internal water compartment andexternal bulk water. Several acceptable methods for forming liposomesare known in the art. Typically, molecules form liposomes in aqueoussolution like dipalmitoyl phosphatidylcholine. Liposomes may beformulated with cholesterol for added stability and may include othermaterials such as neutral lipids, and surface modifiers such aspositively or negatively charged compounds. The preferred liposomes aresmall unilamellar-bilayered spherical shells.

A method for encapsulating hemoglobin in a liposome is also known (SeeFarmer et. al., U.S. Pat. No. 4,911,921). For the purpose of thisinvention, a number of approaches may be used to introduce thenitroxide-based oxygen detoxification function to a solution of liposomeencapsulated hemoglobin. For example, it is possible to usenitroxide-labelled native hemoglobin, or a nitroxide-labelled stabilizedhemoglobin as disclosed above, as the starting material and thenperforming the process of liposome encapsulation of thenitroxide-labelled hemoglobin by known techniques. In the presentembodiment, purified hemoglobin may also be coencapsulated with amembrane impermeable nitroxide such as TEMPO-choline chloride disclosedfor a spin membrane immunoassay in Hsia et. al. U.S. Pat. No. 4,235,792.

Also, any purified hemoglobin may be encapsulated with a liposomecomprised of nitroxide-labelled fatty acids (e.g., 7-DOXYL-stearate,12-DOXYL-stearic acid, and 16-DOXYL-stearate), cholestane, an analogueof cholesterol (e.g., 3-DOXYL-cholestane), or phospholipid (e.g.,12-DOXYL-stearate-labelled phosphatidylcholine). The preparation ofhemoglobin encapsulated in a liposome comprised of 3-DOXYL-cholestanelabelled may be prepared by a method analogous to that described inTabushi et. al., (J. Am. Chem. Soc. 106:219 (1984)). A 5 ml chloroformsolution containing lipid compositions, including DOXYL labelled stearicacid and/or cholestane, as specified below were first dried in a streamof nitrogen to remove the solvent. Next, the residues were dried invacuo and the resulting film was suspended in 2 ml of hemoglobin (24g/dl) in a Lactated Ringers solution. The lipid concentration in thedispersion is 100 mM. The liposome encapsulated hemoglobin is thenrotated and incubated preferably at 37° C. until all lipids aredispersed to form multilamellar vesicles. The resulting solutioncontaining multilamellar liposome encapsulated hemoglobin and freeunencapsulated hemoglobin is then forced through a microfluidizer toform 0.2 micron liposomes according to the procedure of Cook et.al. (SeeU.S. Pat. No. 4,533,254). The molar ratio of dipalmitoylphosphatidylcoline: cholesterol: dipalmitidyl phosphatidic acid:3-DOXYL-cholestane in the liposome is 0.5:0.4:0.02:0.07. The resonancespectrum of the resulting 3-DOXYL-cholestane labelledliposome-encapsulated hemoglobin is shown in FIG. 10A. In thisconfiguration, the nitroxide is intercalated into the liposome membraneand can be found at both the inner and outer surface of the lipidbilayer water interface. Substituting the 3-DOXYL-cholestane with16-DOXYL-stearic acid in the lipid composition shown in FIG. 10A resultsin an electron resonance spectrum shown in FIG. 10B. The mobility of thenitroxide as reflected from the resonance spectrum is consistent withthe interpretation that the DOXYL-moiety of the stearic acid is locatedpredominately in the hydrophobic interior of the lipid bilayer. With theaddition of both the 3-DOXYL-cholestane and 16-DOXYL-stearate to thelipid composition at the same molar ratio, the resonance spectrum of thedouble nitroxide-labelled liposome encapsulated hemoglobin is shown inFIG. 10C. The resonance spectrum of FIG. 10C is a composite of FIGS. 10Aand 10B because the nitroxides in this embodiment are located at boththe membrane-water interface and its hydrophobic lipid bilayer interior.By placing the nitroxide in both locations, this embodiment provides theoxygen detoxification function at both the lipid bilayer hydrophobicinterior and the membrane-water interface thus providing the addedbenefit of an additional reserve of oxygen-detoxification capacity forthe encapsulated hemoglobin.

EXAMPLE FIVE Nitroxide-Labelled Conjugated Hemoglobin

A physiologically compatible solution of conjugated hemoglobin isproduced by forming a conjugate of hemoglobin and a biocompatiblemacromolecule used as a plasma expander. Plasma expanders, such asdextran (Dx), polyoxyethylene (POE), hydroxylethyl starch (HES), areused to increase the circulation half life of hemoglobin in the body. Inthis state, the hemoglobin molecules together with the biocompatiblemacromolecule are collectively referred to as a hemoglobin conjugate.There are a number of convenient methods to incorporate a nitroxide intoa hemoglobin conjugate. For example, one may simply substitute thehemoglobin to be conjugated with a nitroxide-labelled hemoglobin such asTEMPO labelled DBBF-Hb. This can be accomplished by substitutinghemoglobin or pyridoxylated hemoglobin with 3-maleimido-PROXYL-DBBF-Hbor 4-(2-bromoacetamido)-TEMPO-DBBF-Hb in the preparation of conjugatedhemoglobin.

4-Amino-TEMPO labelled dextran conjugated hemoglobin is prepared inaccord with the procedure described by Tam et. al. (Proc. Natl. Acad.Sci., 73:2128 (1976)). Initially, an 8% hemoglobin solution in 0.15MNaCl and 5 mM phosphate buffer, pH 7.4 is conjugated toperiodate-oxidized dextran to form a Schiff-base intermediate. Twentymolar equivalents of 4-amino-TEMPO is added to hemoglobin to form theSchiff-base between the nitroxide and the remaining reactive aldehydegroups on the dextran. After a 30 minute of incubation at 4° C., a 50molar equivalent of dimethylamine borane in water is added. The solutionis incubated for a further 2 hours at 4° C. Afterwards, the solution isdialyzed, reconstituted with Lactate Ringers buffer and sterile filteredwith Filtron membrane filtration units (Filtron Technology Co.). Theelectron spin resonance spectrum of the 4-amino-TEMPO labelleddextran-conjugated hemoglobin is a sharp asymmetric triplet reflecting ahigh degree of motional freedom (See FIG. 11). The increased mobility ofthe TEMPO covalently attached to the Dextran is consistent with thenitroxide linked to a flexible polysaccharide dextran chain as comparedto that of a tightly folded hemoglobin molecule (See FIGS. 3A and 3B).Thus, resonance spectrum in FIG. 11 demonstrates that a novelnitroxide-labelled dextran conjugated hemoglobin has been prepared.

EXAMPLE SIX Nitroxide-Labelled Albumin

A preferred embodiment of this invention is the use ofnitroxide-labelled biocompatible macromolecules in connection with lowmolecular-weight, membrane permeable-nitroxides to provide sustainedantioxidant activity in vivo. Preferably, the nitroxide is labelled to abiocompatible protein, as used, however, the term "protein" includesfragments thereof, by labelling at a large portion of the amino groupsof the protein. Additionally, labelling at the disulfide bonds increasesthe molar ratio of nitroxide to protein. By so labelling the protein, anacidic microenvironment is created which enhances the interactionbetween the free nitroxide and the macromolecule-bound nitroxide,facilitating election spin transfer due to the differential stabilitiesof the species. In the case of albumin, it is also possible tocovalently bind a nitroxide to the primary bilirubin binding site of thealbumin by activating the TOPS. By selecting the binding site on themacromolecule, the reactivity of the nitroxide is modified and thismodification can be used to alter the catalytic activity of thecompound.

An example of such a desirable biocompatible macromolecules is humanserum albumin (HSA).

Serum albumin is a plasma protein with multiple ligand-binding sites andis the transport protein for many ligands in the blood. Nitroxides canbind specifically to human serum albumin at a number of specific ligandbinding sites, or non-specifically. Nitroxide-albumin may be used eitheralone or in combination with a low molecular weight nitroxide compound,e.g., TEMPOL. Nitroxide-labelled albumin is also available as an"improved" version of albumin (i.e., improved by having antioxidantactivity) with utility in any application where albumin is nowconventionally used, including as a parenteral colloid solution, inbiomaterials, in biocompatible surface coatings, etc.

The albumin may be obtained from plasma or may be produced byrecombinant genetic means. HSA may be used in a variety of forms,including monomers (normal plasma form), homodimers, oligomers, andaggregates (microspheres). Additionally, albumin may be treated withpolyethyleneglycol to reduce its immunogenicity. Specific labelling ofthe albumin with a nitroxide may be achieved at several binding sites,including bilirubin, FFA, indole, or Cu⁺⁺ binding site by usingnitroxide compounds which have been activated in order to confer uponthem binding specificity of the relevant site on the protein. Apreferred example is 2,2,6,6-tetramethyl-1-oxyl-4-piperidylidenesuccinate (TOPS) nitroxide covalently bound to the primarybilirubin-binding site of HSA. Non-specific labelling of albumin may beachieved at approximately 50 accessible amino groups. Temperature andchemical treatment of the albumin permits increasing the molar ratio ofnitroxide to albumin. Using native albumin, molar ratios above 7 and upto 60 can be achieved. Using nitroxide-labelled albumin up to 60 moles,a molar ratio of nitroxide to albumin may be accomplished up to 95 maybe achieved by labelling the 35 potential sulfhydrol groups of thealbumin with additional nitroxides.

To demonstrate the regeneration of the active nitroxide in vivo,4-hydroxyl-2,2,6,6-tetramethyl-piperdine-N-oxyl (TEMPOL) in phosphatebuffered saline was injected into the tail vein of an anesthetized mouse(body weight 40 g) as a control. The tip of the tail was inserted intothe sample tube of an electron spin resonance (ESR) spectrometer. Themouse tail displayed no esr signal before the injection. After theinjection of 0.5 ml of 6 mM TEMPOL, an esr signal was detected andobserved to decay rapidly, as seen in three successive scans of thespectrum at 30 second intervals (see FIG. 18). This result indicatesthat the TEMPOL was reduced to its esr-silent, and catalyticallyinactive, N-hydroxylamine (R-NOH) derivative (TEMPOL-H or TPH). Tomeasure the plasma half-life of TEMPOL in the mouse tail, the intensityof the high-field peak was monitored continuously for 8 minutes (FIG.19). After 8 minutes, a repeat injection of TEMPOL was made. The maximumpeak intensity, which corresponds to TEMPOL (TPL) concentration, wasattained in the mouse tail in approximately 10 seconds, followed by arapid decay to base line, resulting in a half-life in vivo of TEMPOL ofapproximately 50 seconds (FIG. 19). The half-life of TEMPOL wasconfirmed by two recording methods: scanning the entire spectrum atintervals and continuous recording of the high-field peak intensity. Theresults from both methods were in agreement.

To prepare the polynitroxide-albumin (PNA), human serum albumin (HSA, 5%solution, Baxter Healthcare Corp.) was allowed to react with 6 molarequivalents of Br-TEMPO (Sigma) at 60° C. for 10 hours with mixing. Theresulting reaction mixture, containing 15 ml of HSA and 165 mg ofBr-TEMPO in a Vacutainer tube, was filter-sterilized with a 0.22 micronfilter and transferred into a 150 ml stirred cell (Stirred Cell Devices)equipped with a 10 kda-cutoff ultrafiltration membrane (FiltronTechnology Corp.). The filtered reaction mixture was washed withRinger's solution (McGaw Inc.) until the filtrate contained less than 1mM of free TEMPO as detected by esr spectroscopy. The bright orangeretentate was concentrated to 25% HSA and again filter-sterilized with a0.22 micron filter into 10 ml sterile vial (Abbott Laboratories) andstored at 4° C. until use. The esr spectrum of the macromolecularpolynitroxide is shown in FIG. 15C.

To increase the molar ratio of nitroxide in a polynitroxide albumin, thedisulfide groups are reduced to sodium thioghycolate in the presence ofurea and an excess of BR-tempo is added to label the broken disulfidebonds. An average of increase in the molar ratio of approximately 10 isachieved by this procedure, which is analogous to the proceduredescribed by Chan et al in "Potential of Albumin Labeled with Nitroxidesas a Contrast Agent for Magnetic Resonance Imaging and Spectroscopy,"Biconjugate Chemistry, 1990, Vol. 1, pages 32-36.

The concentration of a polynitroxide albumin solution is adjusted to17.5 mg/ml in a flask. This solution is diluted with urea to 8M andstirred until dissolved. The pH is adjusted to 8.2 by addition of 2%sodium carbonate. The ph-adjusted solution is degassed completely for 15minutes. Then flashed with argon gas. In another flask, a 3M solution ofthioglycolate is prepared, degassed, and flashed with argon gas. Thethioglycolate solution is added to the polynitroxide albumin to a finalconcentration of 0.3M.

The resulting solution is degassed and flashed with argon gas to mixtureand allowed to stand in the dark under argon gas for 20 hours at roomtemperature, followed by dialyzation against 3L of degassedphosphate-buffer saline pH 8.4 (adjusted by 2% sodium carbonate) for 5hours at room temperature under argon gas. An excess of Br-tempo isadded and the mixture stirred for an additional 24 hours at roomtemperature under argon gas.

The final solution is dialyzed against PBS pH 7.4 for 5 hours to removeunreacted nitroxide. EPR spectroscopy may be used to determine spindensity and protein concentration may be determined by Bruiet method.

Typical results are set forth below for the average of three lots':

1. Protein concentration:

Amino group labelled polynitroxide albumin

53.0 mg/ml=0.78 mM

Amino plus disulfide

13.9 mg/ml=0.20 mM

2. Spin density:

Amino group labelled polynitroxide albumin (PNA)

33 mM

Amino plus disulfide

10.5 mM

3. Calculation of molar ratio of nitroxides bound to protein.

Amino group labelled polynitroxide albumin

33 mM/0.78 mM=42 nitroxides/albumin

Amino plus disulfide

10.5 mM/0.20 mM=52 nitroxides/albumin

The molar ratio is increased by ten in this example.

To demonstrate that a polynitroxide-labelled macromolecule enhances thein vivo activity of the low molecular weight membrane-permeablenitroxide, human serum albumin was covalently labelled with4-(2-bromoacetamido)-TEMPO (BR-TEMPO) and infused after a dose of TEMPOLhad been administered and had been observed to have been converted toits reduced state.

Two hours after the injection of TEMPOL as in the above Example, themouse tail showed no detectable esr signal. When 0.2 ml of thepolynitroxide albumin was injected via the tail vein, the TEMPOL signalreappeared within 4 minutes. The TEMPOL signal intensity persisted formore than two hours, with a half-life of approximately 40 minutes (alsosee FIG. 18). The TEMPOL signal was distinguished from the polynitroxidealbumin signal by their distinctive spectral profiles. The TEMPOL signalwas measured as the intensity of its characteristic high-field peak. Anitroxide to albumin molar ratio of 27 displays the same reactivity.

The possibility that the esr signal detected after injection of thepolynitroxide albumin was due solely to nitroxide on the macromoleculedisassociated from the macromolecule, was ruled out by the followingexperiment. The experiment was performed as above, but using ¹⁵ N!-TEMPOL and albumin- ¹⁴ N!-TEMPOL. The different nitrogen speciesprovide a method of discriminating the esr signals of the free and themacromolecular nitroxides. The results (FIG. 18) confirm that the esrsignal from the regenerated nitroxide is derived from the ¹⁵ N isotopeand, therefore, that the antioxidant activity of the low molecularweight membrane permeable ¹⁵ N!-TEMPOL has been regenerated followingthe addition of the macromolecular polynitroxide.

EXAMPLE SEVEN Nitroxide-Labelled Immunoglobulin

As in the above embodiments, certain nitroxides have been shown to havevery short plasma half-life when injected intravenously. Due to thedesire to have an antioxidant enzyme mimic with a long plasma half-life,a nitroxide compound may be attached to an immunoglobulin to providelong-lasting antioxidant-enzyme mimic activity.

Immunoglobulins are a class of plasma proteins produced in the B-cellsof the immune system and which are characterized by two specific ligandbinding sites (the antigen-binding sites). Nitroxides have been used inthe past as probes in research on hapten-binding specificity andaffinity of immunoglobulins during the primary and secondary immuneresponse.

As with the above-embodiment describing nitroxide-labelled albumin, thenitroxide-labelling technology demonstrated above in the example ofnitroxide-HBOC is readily applied to the production ofnitroxide-labelled immunoglobulins. Immunoglobulins after the advantageof specific binding and long circulatory half-life such that theenzyme-mimic activity of the compounds of this invention can be targetedto specific tissues and have prolonged activity.

Nitroxide-labelled immunoglobulins may be used in vivo to provideprotection against cellular damage by reactive oxygen species.Nitroxide-labelled immunoglobulin may be used either alone or incombination with a low molecular weight nitroxide compound to provideextended antioxidant activity with an extended plasma half-life.

Nitroxide-labelled immunoglobulin may be prepared by specific labellingof the immunoglobulin itself or by covalently labelling at ahapten-binding site. To avoid clearance of the nitroxide-labelledimmunoglobulin as part of the body's natural immune response, one mayuse immunoglobulin fragments, for example, (Fab)₂ produced by cleavingthe immunoglobulin according to known techniques withnon-specific-labelling, a preferred molar ratio ofnitroxide:immunoglobulin is up to 60:1.

Nitroxide-labelled immunoglobulins are a preferred species for use totarget the enzyme-mimic effect of a particular location. For examples byselecting antibodies specific to an antigen implicated in inflammationor other such pathology. The image enhancing and therapeutic benefits ofthis invention can be targeted at a particular site.

EXAMPLE EIGHT Imaging of Biological Structures and Free RadicalReactions by EPR

Under most circumstances, free radical reactions occur so rapidly thatEPR imaging (ERI) is difficult. However, due to the presence of stablefree radicals, nitroxides are detectable by electron paramagneticresonance spectroscopy. With the development of advanced imaginginstrumentation images of intact biological tissues and organs areavailable based on a measurement of free radical concentration.Biocompatible nitroxides are candidates for image-enhancing agents.

Because nitroxides are reduced in vivo to inactive derivatives within afew minutes of administration, their utility is limited. Pursuant tothis invention, active nitroxide levels in the body may be maintainedfor a prolonged period of time allowing both improved image contrast andlonger signal persistence than seen with low molecular weight membranepermeable nitroxides alone.

Electron paramagnetic resonance (EPR) spectroscopy is a technique forobserving the behavior of free radicals by detecting changes in theenergy state of unpaired electrons in the presence of a magnetic field.The technique is specific for free radicals because only unpairedelectrons are detected. Using available apparatus that measure electronparamagnetic resonance, a real-time image of a macroscopic object,including living tissue can be obtained. EPR imaging (ERI) provides thecapability to obtain multidimensional images (including spectral-spatialimages) for diagnosis or research.

Electron paramagnetic resonance imaging (ERI) with nitroxide contrastagents is in principle a valuable method for medical imaging,particularly the imaging of ischemic tissue. However, the development ofthis technology has been limited by the fact that nitroxides are rapidlyreduced in vivo to non-paramagnetic species.

The application for extrinsically introduced nitroxide has demonstratedutility as a relatively low resolution in vivo EPR imaging agent. (L. J.Berliner, "Applications of In Vivo EPR," pp. 292-304 in EPR IMAGING ANDIN VIVO EPR, G. R. Eaton, S. S. Eaton, K. Ohno, editors; CRC Press(1991). Importantly, Subramanian and his collaborators constructed aradio frequency fourier transform EPR spectrometer for detecting freeradical species and for in vivo imaging. J. Bourg et al., J. Mag. Res.B102, 112-115 (1993).

A polynitroxide albumin (PNA) prepared pursuant to this invention, isadministered which distributes in the vascular and other extracellularspaces. Although the range of concentrations of the compositions of thisinvention may vary. A preferred range is 5-25 g labelled PNA perdeciliter and 0.1 to 200 mM TEMPOL. The antioxidant activities anddetectability by electron paramagnetic resonance (EPR) spectroscopy andare useful for this purpose alone. Additionally, when administered withmodest doses of small molecular weight, membrane permeable nitroxides,the nitroxide is maintained in an active free radical state in the bodyfor a prolonged period of time.

A preferred formulation for ERI imaging is human serum albumin (1.0 to25.0 g/dl) covalently labelled with a high molar ratio of nitroxide (7to 95 nitroxide to albumin). As noted elsewhere herein, thepolynitroxide albumin (PNA) can accept an unpaired electron from thehydroxylamine form of the nitroxide (e.g., TPH) regenerating theactivity of the nitroxide to its free radical state (e.g., TPL). Afundamental advantage of this multi-component system is that while themacromolecule or species remains in the extracellular space, the small,membrane-permeable nitroxide, and its reduced (hydroxylamine)derivative, distribute freely between the intracellular andextracellular spaces. This creates a cycle in which nitroxide freeradical can be detected within the cell prior to being reduced, followedby regeneration by the macromolecular (extracellular) species. Thus, adesired concentration of a spectroscopically detectable nitroxide can bemaintained in vivo for a prolonged period of time.

Extending the short half-life of nitroxides in vivo helps overcome amajor obstacle in the development of a nitroxide-based imaging methodand medical therapy. With regard to imaging, the EPR Laboratory at theJohns Hopkins University has been developing a continuous-wave ERIinstrument utilizing nitroxide as contrast agent to study the course ofcardiac ischemia and reperfusion. However, the limited half-life ofnitroxide alone has meant that the reperfusion phase cannot be studied.In these studies, the three-dimensional spectral-spatial EPR imaging ofnitroxide in the rat heart suffer from the rapid decay of free radicalsignal due to nitroxide reduction. Although a cross-sectional transverse2-D spatial EPR image of the rat heart has been reconstructed from a 3-Dspectral-spatial data, this was based on an EPR signal which wasdecaying continuously during the 12-minute acquisition period. Thedifferent rates of nitroxide reduction in the epicardium, midmyocardium,and endocardium, as a function of the duration of ischemia, furtherreduces the definition of the cross-sectional image of the heart.

Pursuant to this invention, the ERI image may be improved. The extendedactive half-life of an in vivo nitroxide permits imaging of thereperfusion phase and provides additional information on the progress ofischemic injury to tissues and organs. The compositions of thisinvention provide a stable nitroxide signal suitable for imaging within10 minutes after administration, and persists for at least approximately2.0 hours. (See FIG. 25). For comparison, the signal from a freenitroxide alone effectively disappears within 20 minutes afteradministration (FIG. 24). With regard to therapeutic utility, theability to safely maintain active nitroxide levels for prolonged periodsof time represents the ability to provide an extended antioxidant effectaiding in the prevention of ischemia/reperfusion injury, pathologicalprocesses where toxic oxygen-derived free radicals are the agents ofcell damage (see FIG. 26 and FIG. 27).

Using methods essentially as described in Kuppusamy et al. Proc. Natl.Act. Sci., USA Vol. 91, pgs. 3388-3392 (1994), the rat heart was imagedusing polynitroxide albumin (PNA) at a concentration of 4 g albumin/dland 2 mM ¹⁵ N-TEMPOL.

Referring to FIG. 24, the signal intensity of ¹⁵ N-TEMPOL in thepresence of polynitroxide albumin, can be seen in the isolated ratheart. The EPR signal was stable with a bi-phasic and gradual decline inintensity. This contrasts with the bi-phasic rapid decline in signalintensity using a low molecular weight membrane permeable nitroxide(TPL) alone (FIG. 24).

In an EPR imaging study, the rat heart was perfused with a solutioncontaining nitroxide with or without polynitroxide albumin, followed bycessation of perfusate flow in order to create ischemia. FIG. 24 showsthe total signal intensity of ¹⁵ N!-TEMPOL in the isolated rat heartduring ischemia in the presence and absence of polynitroxide albumin. Itcan be seen the signal intensity undergoes a biphasic decay and that thepresence of polynitroxide albumin greatly slows the decay. By thusstabilizing the TEMPOL signal, polynitroxide albumin allows high-qualityEPR images to be obtained over a prolonged period of time. Referring toFIGS. 22-23, a three-dimensional EPR image of the ischemic heart canstill be obtained after 156 minutes of global ischemia. FIG. 22A shows acut-out view of the image. As illustrated in FIG. 23, thethree-dimensional image can also be viewed in cross-section. This showsdifferential distribution of TEMPOL signal within the ischemic heart;this is quantitated in FIG. 28. FIG. 25 shows a series of images such asin FIG. 23, acquired at a series of successive time points during 125minutes of global ischemia. This illustrates that the presence of PNAallows much better imaging than is possible with TEMPOL alone, in termsboth of resolution and signal persistence.

A particular advantage of this invention is the ability to localize theimage enhancement by selecting compositions which are selectivelydistributed in the structure of interest and/or by selecting a means ofadministration which facilitates image enhancement of a particularstructure or system. For example, image enhancement of thecardiovascular system can be provided by intravenous infusion of apolynitroxide macromolecule having a lengthy plasma half-life and,preferably, an intravenous infusion of low molecular-weight membranepermeable nitroxide. Alternatively, to enhance an image of thegastro-intestinal tract, one of these compounds may be administeredorally. Similarly, an image enhancement of a discrete region of the skinmay be obtained by a topical administration of a polynitroxidemacromolecule suspended in a suitable carrier, combined with oral orintravenous administration of an additional nitroxide species.Alternatively, depending on the application, multiple nitroxide-basedcompositions pursuant to this invention could be dermally administered.

EXAMPLE NINE Radiation Protection

Living organisms which are exposed to ionizing radiation suffer harmfuleffects which can be fatal with high doses of radiation. Recent evidencesuggest that radiation causes cellular injury through damage to DNA. Ofthe total damage to DNA, as much as 80% may result fromradiation-induced water-derived free radicals and secondary carbon-basedradicals. The Department of Defense has had screened over 40,000aminothiol compounds looking for an in vivo radiation protector.Although one agent (WR-2721) showed selective radioprotective effects,WR-2721 failed to exhibit radiation protection in human clinical trials.A nitroxide compound (e.g., TPL) was a non-thiol radiation protectiveagent, but as noted herein, (TPL) has a very short in vivo half-life.The other compounds were macromolecules of natural origin (Superoxidedismutase, IL-1, and GM-CSF). However, due to their molecular size, eachof these has limited capability to provide intracellular free radicalscavenging.

The National Cancer Institute attempted to use the nitroxide compoundTempol as a radioprotective agent to allow greater dosage levels ofradiation treatment of cancer patients. The researchers quickly foundthe low molecular weight nitroxide was quickly reduced to an inactiveform and safe dosages could not be administered.

Based on the invention disclosed herein, nitroxides bound tomacromolecular compounds as enzyme mimics can be used together with lowmolecular-weight nitroxides to detoxify oxygen radicals in the vascularspace by interacting with membrane-permeable nitroxide compounds todetoxify free radicals inside cells. Extending the duration of theradioprotective effects pursuant to this invention allows the use ofnitroxide compounds to protect against the harmful effects of controlledradiation in medical applications such as cancer therapy, and inaccidental exposure to harmful radioactive sources.

Based on a radiation dosage scale developed by the National CancerInstitute, chinese hamster cell cultures are exposed to ionizingradiation in the presence of radioprotective chemical agents. FIG. 16shows the survival rate of Chinese hamster V79 Cells at 12 Gray ofradiation. The control, macromolecular bound nitroxide (PNA), andreduced nitroxide (TPH) show similar survival rates. However, TPHpremixed with PNA results in the conversion to a radioprotective TPL(see FIG. 17) which enhances the survival of the V79 cells (see FIG.16).

Low molecular weight, membrane-permeable nitroxides e.g., TPL have beendemonstrated to provide radiation protection in vivo in C3H mice. Inthese studies, the maximal tolerated dose of TPL administeredintraperitoneally was found to be 275 mg/kg, which resulted in maximalTPL levels (˜150 μg/ml) in whole blood 5-10 minutes after injection.Mice were exposed to whole body radiation in the absence or presence ofTPL (275 mg/kg) 5-10 minutes after administration. The dose of radiationat which 50% of TPL treated mice die within 30 days was 9.97 Gray,versus 7.84 Gray for control mice.

Because the radioprotectant effect of TPL is derived from the reactivityof the unpaired electron, when TPL is reduced to hydroxylamine by losingits unpaired electron, it become inactive. The effective radioprotectiveagent, pursuant to this invention, maintains in vivo a therapeuticconcentration of TPL in its active (free radical) state while overcomingthe fact that when TPL is administered alone, the dosage required tomaintain therapeutic levels is high and is toxic.

FIG. 19 shows that the maximum plasma level of TPL after intraperitonealinjection of 275 mg/kg of TPL (⋄) 2 mM TPL alone by intravenousadministration (□), and TPH 100 mg/kg+PNA 1 ml (◯). The results showthat the maximum plasma level of TPL is approximately one-fifth of thatobserved after the intraperitoneal injection of approximately 100 mg/kgof TEMPOL in the presence of PNA (0.5 ml/mouse at albumin concentrationof 20 g/dl and 42 moles TPL per mole of albumin). Therefore, the plasmalevel of TPL is enhanced by greater than tenfold in the presence of PNA.This enhanced plasma level of TPL influences the intracellular levels ofTPL which are responsible for radiation protection at the cellular andnuclear levels.

This enhance protection is demonstrated in full body irradiation basedon a 30-day survival model in mice. FIG. 20 shows enhancement ofradiation protection by the addition of PNA at a constant TPLconcentration (200 mg/kg).

The results show that TPL in the presence of PNA has a profoundradioprotective effect. Eight out of ten (80%) mice survived the 10 Graylethal radiation as compared to one out of ten (10%) with TPL alone. Ina control experiment, without TPL or with PNA alone, all mice die on orabout day 15. Therefore, the membrane impermeable PNA shows no radiationprotective effect and does not protect against radiation damage at theintracellular level.

Referring to FIG. 21, the experimental data shows that PNA enablesreduction in the TPL dose to achieve similar radiation protection. Inthis experiment, all ten mice died on day 15 when PNA (0.5 ml/mouse) wasused alone. At one quarter the dose of TPL used in FIG. 20, the TPLconcentration is reduced from 200 mg/kg to 50 mg/kg, the presence of PNAwas able to protect two out of ten mice from lethal radiation (10 Gray).These results demonstrate that PNA can be used to reduce the dosage ofTPL by a factor of four to achieve the same or better radiationprotection.

Due to the ability to provide a systemic or physically localizedradioprotectant effect, the use of the compositions of this invention isparticularly advantageous in protecting a patient undergoing therapeuticradiation treatments. For example, a systemic or localized topicaladministration of a membrane permeable nitroxide can be combined with atopical administration of a polynitroxide albumin at the site where aradiation flux enters the body.

In one particular application, a topical ointment containing apolynitroxide albumin is applied to the scalp of a patient undergoingtreatment for a tumor of the cranium. Concurrently therewith, a membranepermeable nitroxide is administered by an appropriate means, includingfor example suspension in the topical ointment. When the radiation doseis applied, the skin and hair follicles will be protected from thecomplete harmful effects of the radiation thereby lessening damage tothe skin and reducing hair loss. An additional example involving braintumors is particularly significant because of the high mortality rateand difficulty in successful surgical therapy. A whole body radiationprotection can be provided by systemic administration of a membranepermeable nitroxide and topical and intravenous administration of apolynitroxide macromolecule with decreased permeability of the tumor. Insuch an example, an increased dose of radiation can be administered dueto the whole body radioprotective effect of the invention. Due toselective permeability, the tumor region is more susceptible toradiation than the surrounding tissue and is thereby treated withgreater efficacy. The selection of a number of variations andmodifications of the above examples is well within the skill of those inthe pertinent art as are other modifications which do not depart fromthe spirit of the invention.

EXAMPLE TEN In Vivo Enzyme Mimic

As noted above, nitroxides (e.g., TEMPOL) have been shown to havecatalytic activity which mimics that of superoxide dismutase (SOD), themetalloenzyme which dismutes superoxide to hydrogen peroxideFurthermore, in biological systems, nitroxides can interact withperoxidases and pseudoperoxidases to achieve an activity mimicking thatof catalase, the enzyme which converts hydrogen peroxide to oxygen.Demonstrated herein is the use of nitroxides to mimic a superoxideoxidase to alleviate oxidative stress associated with metabolism ofoxygen carriers. The biological effects of such activity derived fromnitroxide-containing compounds include contributing to protectionagainst cytotoxicity of reactive oxygen species by reducing oxidativestress. Nitroxides, when administered in vivo pursuant to thisinvention, display additional complex antioxidant enzyme-mimeticactivities.

As noted above, when injected intravenously, TEMPOL has been shown tohave very short plasma half-life. Due to its molecular size and chargecharacteristics, it readily leaves the vascular space. In many medicalapplications, it may be desirable to have an enzyme mimic which persistsin the vascular space. This is achieved pursuant to this invention, byattaching a nitroxide compound to a macromolecule, such as hemoglobinand albumin, which is biologically safe and has a desirable plasmahalf-life.

A membrane-permeable nitroxide such as TPL, in its free radical statehas been shown to have enzyme-mimic activity both in vitro and in vivo.However, in vivo, primarily in the intracellular space, it is rapidlyreduced to its inactive hydroxylamine derivative (TPH) by bioreducingagents such as NADH. Previously, the reduction of the active TPL to theinactive TPH has been essentially irreversible on a stoichiometricbasis. Thus, its effectiveness as a therapeutic and diagnostic tool islimited.

Pursuant to this invention, a multi-component nitroxide-containingcomposition has, as a first component, the membrane-permeable nitroxidewhich exists in a dynamic equilibrium between TPL (active) and TPH(inactive).

In vivo, the inactive TPH predominates (>90%). Both molecular species(TPL and TPH) readily cross the cell membrane and distribute into theintracellular and extracellular spaces.

A second component is a membrane-impermeable, macromolecularpolynitroxide which distributes in the extracellular space,predominantly in the vascular space. The first and second componentsexhibit another enzyme mimetic function previously unknown in vivo, thatof a synthetic reduced-nitroxide oxidase. For example, the polynitroxidealbumin described herein as part of a multi-component system acts as areduced-nitroxide oxidase by oxidizing TPH and TPL via a spin-transferreaction. Thus, the macromolecular polynitroxide albumin acts as anenzyme mimic shifting the TPL/TPH equilibrium up to ˜90% TPL in both theintra and extra-cellular spaces. This enzyme-mimic function isparticularly useful where a high dose of TPL necessary to produce therequisite level of protection from radiation, ischemia, etc. would betoxic to the cells by overwhelming their cellular redox machinery.

For example, in the example of a dose of gamma radiation, when the dosebecomes elevated, the quantity of low molecular weight, membranepermeable nitroxide necessary to provide meaningful radioprotectiveeffects can become so large that the cells redox state is disrupted,thereby resulting in toxicity.

To overcome the toxicity hurdle, the multi-component system of thisinvention regenerates the reduced TPH to TPL. This system can be used inany application where an active nitroxide is desirable in vivo.

The EPR spectra of TPL TPH and a polynitroxide albumin is shown in FIGS.15A, 15B, and 15C, respectively. Demonstration of a reduced nitroxideoxidase activity is shown by the reoxidation (spin-transfer) from TPH(EPR silent FIG. 15B) to the macromolecular polynitroxide (FIG. 15C) toand its conversion to TPL (EPR active) (FIG. 15A) is carried out asfollows: (1) equimolar ratios of TPH to a macromolecular polynitroxideare incubated at room temperature for 30 minutes; (2) the reactionmixture is centrifuged through a 10 kd cut-off membrane; and (3) The EPRspectrum of the filtrate is recorded and shown in FIG. 15B. Thequantitative-conversion of TPH to TPL is shown in FIG. 17.

A synthetic reduced nitroxide oxidase (polynitroxide albumin) isprepared by allowing human serum albumin (HSA, 25% Baxter Healthcare) isreacted with 40 molar equivalents of 4-(2-bromoaceamido)-TEMPO orBr-TEMPO at 60° C. for ten hours with mixing. The resulting mixture,containing 15 ml of HSA and 165 mg or Br-TEMPO in a vacutainer tube, issterilized with a 0.22 micron filter and transferred into a 150 mlstirred cell equipped with a 10 kd cutoff membrane (Filtron TechnologyCorp.). The filtered reaction mixture is washed with Ringers solutionuntil the filtrate contains less than 1 uM of Free TEMPO as detected byESR spectroscopy. The bright orange colored retenate is concentrated to25% HSA and again sterile-filtered into a 10 ml vial and stored at 4° C.until used. To demonstrate the in vivo enzyme-like conversion of TPH toTPL, the ¹⁵ N stable isotope analogue of TPL is injected into the tailvein of a cannulated mouse and the EPR signal is directly monitored inthe tail. The direct intravenous injection of a 0.5 ml of TPL (40 mM)solution in the anesthetized mouse demonstrates that the plasmahalf-life of TPL is approximately 2 minutes. Referring to FIG. 18, witha follow-on injection (˜30 minutes later) of a mixture comprising amacromolecule polynitroxide and a stable isotope ¹⁵ N TPL, a biphasicchange in the peak intensity of ¹⁵ N TPL exists. Initially, a decreasein ¹⁵ N TPL signal intensity is attributed to the diffusion of TPL outof the-vascular space followed by its intra-cellular reduction. Thisrate of diffusion is initially faster than the rate of reoxidation ofthe TPH to TPL based on the slower re-appearance of the ¹⁵ N TPL signalintensity in FIG. 18. Although the reoxidation of TPH to TPL is slowerthan the initial diffusion/reduction rate, it is faster than the steadystate intracellular reduction rate of TPL. Thus, the reappearance of theTPL signal shown in FIG. 18 detects the reoxidation of TPH to TPLthereby demonstrating a synthetically produced reduced nitroxide oxidaseactivity in vivo in mice.

EXAMPLE ELEVEN Ischemia and Reperfusion Injury

As noted above, nitroxide-containing compounds can be used in medicalimaging. A particularly useful application is in obtaining images ofischemic tissues in the heart and elsewhere, because valuableinformation regarding oxygen metabolism and reperfusion injury can beobtained. However, the rapid reduction of free nitroxides in vivo limitsthe utility of free nitroxides in this application. However, pursuant tothis invention, it is possible to enhance the imaging capability tospatially resolve ischemic tissue in the heart, to monitor thedevelopment of myocardial ischemia, to study the development of themyocardial reperfusion phase, and to observe in real time the hypoxicstate of tissues or organs. Referring to FIGS. 22, 23, and 25, ERIimages of an isolated rat heart infused with ¹⁵ N-TEMPOL and apolynitroxide albumin are shown.

In FIG. 24, the intensity of the EPR signal is shown as a function ofthe duration of ischemia. In the lower curve, 2 mM of TEMPOL is infusedinto an ischemic heart. The upper curve traces the original intensity of2 mM TEMPOL together with a polynitroxide albumin (4 g/dl of albumin at42 moles of TEMPOL per mole of albumin). The data demonstrate that thesignal intensity is substantially greater, and is maintained, when thecomposition of this invention is used. FIG. 25 shows the viability ofimaging at ischemic tissue from 3-D spatial images. The progression ofimages traces the progress of ischemia over approximately 125 minutes.

Apart from demonstrating diagnostic utility, the polynitroxide albuminand TEMPOL combination protects the heart from ischemic reperfusioninjury. FIG. 26 shows that a nitroxide alone in combination with PNAdoes not affect the recovery of coronary flow. However, FIG. 27 showsthe substantially improved recovery of RPP (rate pressure product)following 30 minutes of global ischemia followed by 45 minutes of bloodflow is only observed in the presence of both. Furthermore, edema of theheart as a result of ischemic/reperfusion injury was prevented.

Referring to FIG. 28, the ¹⁵ N-TPL concentration in various anatomicalregions of the ischemic heart are elevated during over 100 minutes ofischemia. Elevation of TPL tissue concentration may contribute to theprotection of cardiac function (FIG. 27) by PNA.

EXAMPLE TWELVE Vasodilatory and Vasoneutral Formulation ofHemoglobulin-based Red Cell Substitute (HRCS)

FIG. 13 shows the effect of the compositions of this invention on thevasoconstrictive effect of hemoglobin-based oxygen carriers (HBOC),specifically DBBF-Hb. This vasoconstriction is demonstrated in consciousrat models by measuring the increase in mean arterial pressure (MAP)when a 10% v/v top load of this solution is infused. Referring to FIG.13, the dotted line indicates the mean arterial pressure as a functionof time following infusion of an HBOC. Pursuant to this invention, thesame PNA and TPL solution used for radiation protection, ERI, andischemia/reperfusion injury protection is shown to possess a broad rangeof enzyme mimic and radical detoxification functions. PNA or TPL, wheninjected alone with HBOC were found to have no antihypertensive effect.Further, PNA (5 g/dl) or TPL (100 mM) alone, 10% v/v top load, inconscious rats produces no significant vasodilatory effect. However, PNA(5%/dl)/TPL (100 mM) as top loaded at 10% v/v produces a significant andsustained vasodilatory effect was observed (FIG. 29). This vasodilatoryeffect coincides with the sustained plasma TPL levels in these rats(FIG. 14). In the absence of PNA, the plasma half-life of TPL in theserats is less than 60 seconds. Therefore, by mixing equal volumes of PNA(5 g/dl)/100 mM TPL with DBBF-Hb 7.8 g/dl and top load a 20% v/v inthese rats a vasoneutral HRCS formulation is produced (FIG. 13). Thehypotensive affect observed in FIG. 29 coincides with the sustainedelevation of TPL (FIG. 14) in the vascular smooth muscles, which preventthe destruction of nitric oxide (i.e., endothelium derived relaxingfactor (EDRF) by superoxide), thus enhancing the vasodilation andlowering the MAP in the rat (FIG. 29). In the case of a vasoneutral HRCSformulation (FIG. 13), the vasoconstrictive and vasodilatory activitiesof the HBOC and PNA/TPL cancelled each other's effect on the nitricoxide levels in vivo. Therefore, this vasoneutral formulation of HRCS isa significant improvement of the HBOC currently in clinical development,based on the global protection of free radical and oxidative stress.

The particular examples set forth herein are instructional and shouldnot be interpreted as limitations on the applications to which those ofordinary skill are able to apply this invention. Modifications and otheruses are available to those skilled in the art which are encompassedwithin the spirit of the invention as defined by the scope of thefollowing claims. All references and publications referred to above arespecifically incorporated by reference.

I claim:
 1. A method to protect an organism from ionizing radiationcomprising:administering a membrane permeable first nitroxide, andadministering a polynitroxide albumin labled at an average molar ratioof between approximately 17 to
 95. 2. The method of claim 1 wherein themembrane permeable nitroxide is selected from the group consisting ofTEMPOL (2,2,6,6-Tetramethylpiperidine-N-oxyl, PROXYL(2,2,5,5-Tetramethypyrrolidine-N-oxyl), or DOXYL(4.4-Dimethyloxazolidine-N-oxyl).
 3. A method to treat a physiologicalcondition using ionizing radiation comprising:administering a membranepermeable first nitroxide, administering a polynitroxide albumin whereinthe molar ratio of nitroxide to albumin is between approximately 17 to95, and exposing the organism to ionizing radiation.
 4. The method ofclaim 3 wherein the membrane permeable nitroxide is selected from thegroup consisting of TEMPOL, DOXYL, or PROXYL and the second nitroxide isa nitroxide-labelled macromolecule selected from the group consisting ofhydroxylethyl starch, albumin, hemoglobulin, liposome, orimmunoglobulin.
 5. The method of claim 3 wherein said exposure toionizing radiation is delivered to a site coincident to the in vivodistribution of the second nitroxide species.
 6. The method of claim 3wherein the polynitroxide albumin is in a carrier suitable for topicalapplication and wherein said administration of said polynitroxidealbumin is by topical application at a site where said organism isexposed to said ionizing radiation.
 7. The method of claim 3 wherein atleast one of said membrane permeable nitroxide or said polynitroxidealbumin is administered intravenously prior to exposing said organism tosaid ionizing radiation.
 8. The method of claim 1 wherein the averagemolar ratio of nitroxide to albumin is between approximately 30 and 95.9. The method of claim 1 wherein the average molar ratio of nitroxide toalbumin is between approximately 42 and
 95. 10. The method of claim 1wherein the average molar ratio of nitroxide to albumin is betweenapproximately 52 and
 95. 11. The method of claim 1 wherein the averagemolar ratio of nitroxide to albumin is between approximately 42 and 52.12. The method of claim 3 wherein the average molar ratio of nitroxideto albumin is between approximately 30 and
 95. 13. The method of claim 3wherein the average molar ratio of nitroxide to albumin is betweenapproximately 42 and
 95. 14. The method of claim 3 wherein the averagemolar ratio of nitroxide to albumin is between approximately 52 and 95.15. The method of claim 3 wherein the average molar ratio of nitroxideto albumin is between approximately 42 and 52.